Genetically Modified Microorganisms

ABSTRACT

The present invention relates to genetically modified microorganisms comprising one or more heterologous nucleic acid molecules together encoding at least three different proteins, each protein comprising an enzymatic domain and a bacterial microcompartment-targeting signal polypeptide, wherein said enzymatic domains each catalyse a different substrate to product conversion in the same metabolic pathway, and wherein said microorganisms are essentially free of bacterial microcompartments (BMCs); and to cell free systems comprising aggregates comprising at least three different proteins, each protein comprising an enzymatic domain and a bacterial microcompartment-targeting signal polypeptide, wherein said enzymatic domains each catalyse a different substrate to product conversion in the same metabolic pathway, and wherein said system does not comprise bacterial microcompartments; and to methods for the production of said microorganisms and cell free systems and their use in methods of producing a product of interest.

The present invention relates to genetically modified microorganisms andtheir use in the production of desired products of metabolic pathways,and particularly in improving the levels of production of said products.In particular the microorganisms of the present invention are modifiedto comprise enzymes of a metabolic pathway for a product of interest,wherein the enzymes are each tagged with a bacterial microcompartment(BMC)-targeting signal peptide, and wherein the microorganism lacksbacterial microcompartments. The present invention also relates tocell-free systems comprising said (BMC)-targeting signal peptide taggedenzymes in the absence of BMCs.

BACKGROUND

Bacterial microcompartments (BMCs) are metabolosomes, i.e. discreteprotein-based organelles in which steps of a particular metabolicpathway occur. BMCs are typically 40 to 200 nm in diameter and consistof a semipermeable proteinaceous outer layer that encases the enzymesthat catalyse steps of a particular metabolic process. Thus, within aBMC, enzymatic activity of a particular metabolic pathway or partthereof is concentrated. The encapsulated environment is ideal for thechanneling of toxic/volatile intermediates.

BMCs are thought to be involved in eight or more metabolic processes.BMCs are widely distributed (in approximately 17% of bacteria in 23different phyla). Multiple BMC types can be found in a single genome. Aparticular BMC type is associated with a particular metabolic process.These include anabolic processes such as carbon dioxide fixation (in thecarboxysome) and catabolic processes such as 1,2-propanediol utilisation(in the Pdu BMC) and ethanolamine utilisation (in the Eut BMC) andcholine degradation (in the Cut BMC).

The first characterized BMC was the carboxysome, which is found incyanobacteria and some chemoautotrophs. In the carboxysome, the enzymescarbonic anhydrase and RuBisCo are retained within the confines of themacromolecular complex to provide an environment for enhanced carbondioxide fixation.

The propanediol utilization (pdu) operon is composed of 23 genes andencodes largely for proteins that form a BMC with a diameter of between100 and 150 nm. Six of the genes (pduABJKUT) encode for shell proteinsthat comprise BMC domains as their structural core and form hexamerictiles, which align together to form the facets and edges of the outercasing of the capsule structure. The vertices of the BMCs are thought tobe formed from the pentameric PduN.

The Pdu BMC shell proteins encapsulate the enzymes for 1,2-propanediolmetabolism, including the diol dehydratase (PduCDE), and the alcohol andaldehyde dehydrogenases (PduP and Q). The metabolosome also housesenzymes for the repair and reactivation of the diol dehydratase (PduG,H) and its coenzyme adenosylcobalamin (PduO, S), The shell of the BMCallows the passage of its substrates, cofactors, and coenzymes into theBMC as well as the exit of the metabolic products. This is likelymediated through the central pores that are formed within the tiles ofthe shell structure. Other proteins are thought to interact with theshell proteins on the external surface of the structure, including PduV,which may help to localize the BMC within the cell.

Recent studies have also revealed targeting sequences that mediateprotein encapsulation within BMCs. Enzymes located within BMCs comprisesuch BMC-targeting signal sequences. Previous studies have demonstratedthat tagging proteins not found naturally within BMCs, e.g. GFP, with aBMC-targeting signal sequence results in the localisation of thoseproteins within BMCs.

In the field of metabolic engineering, a number of attempts have beenmade to target metabolic pathways of interest to BMCs. The reasoning forsuch attempts is that pathway encapsulation within a BMC would permitincreased flux through the pathway, control of molecules that enter andexit the BMCs, sequestration of intermediates, concentration ofreagents, optimisation of reaction environment, etc.

Through heterologous expression, empty Pdu BMCs have been successfullyexpressed in cells that lack them in wild-type form. It was found thatsix proteins PduA, B, B′, J, K and N were necessary and sufficient forBMC formation in E. coli. The absence of PduU and PduT did not preventshell formation, even though they are known to be shell proteins. InSalmonella, it has been found that PduM is required, but PduA isdispensable.

Other groups have successfully targeted non-native proteins to BMCs. Thepresent inventors have previously demonstrated the usefulness of BMCsand targeting sequences in the formation of functional bioreactors forthe production of ethanol.

The BMC-targeting sequence of PduP or PduD was fused to the enzymes ofthe ethanol production pathway: pyruvate decarboxylase and alcoholdehydrogenase, and the tagged enzymes were co-expressed with BMC shellproteins in a bacterial cell that does not naturally produce BMCs.Co-production of tagged-enzymes for ethanol formation and the BMC shellproteins resulted in significantly more ethanol in comparison to strainswith cytoplasmic (untagged) enzymes.

US 2012/0210459 discloses various means for designing and implementingBMCs for customizing metabolism in various organisms. Various sequencescomprising BMCs are disclosed and the application teaches the expressionof said sequences in organisms that do not naturally comprise BMCs. Thisdocument teaches co-expression of said BMCs with enzymes of interest,optionally with a BMC targeting signal peptide linked thereto. US2013/0133102 discloses a variety of known and predicted BMC-targetingsequences.

SUMMARY

Surprisingly, the present inventors have now found that increased levelsof a product of interest can be obtained using microorganisms thatcomprise polypeptides having enzymatic domains and BMC-targeting signalsequences, wherein the enzymatic domains catalyse steps of the samemetabolic pathway for the production of said product of interest, butwherein the cell lacks the ability to produce BMCs.

In one aspect, the present invention provides a genetically modifiedmicroorganism comprising one or more heterologous nucleic acid moleculestogether encoding at least three different proteins, each proteincomprising an enzymatic domain and a bacterialmicrocompartment-targeting signal polypeptide, wherein said enzymaticdomains each catalyse a different substrate to product conversion in thesame metabolic pathway, and wherein said microorganism is essentiallyfree of bacterial microcompartments.

Alternatively viewed, the present invention provides a geneticallymodified microorganism comprising at least three different recombinantproteins, each protein comprising an enzymatic domain and a bacterialmicrocompartment-targeting signal polypeptide, wherein said enzymaticdomains each catalyse a different substrate to product conversion in thesame metabolic pathway, and wherein said microorganism is essentiallyfree of bacterial microcompartments.

Without wishing to be bound by theory, the inventors believe that theBMC-targeting signal polypeptides mediate aggregation of said proteins.The result of the proteins of the invention each comprising an enzymaticdomain and a BMC-targeting signal polypeptide is the aggregation ofproteins comprising said enzymatic domains. Although aggregation istypically an undesirable occurrence in protein expression systems, theinventors have determined that in the context of multi-step metabolicpathways, it is surprisingly advantageous. As a result of theaggregation, enzymatic activity is spatially concentrated and there canbe rapid channeling of the product of one enzymatically catalysedmetabolic step to the active site of a second enzymatic domain thatcatalyses a subsequent step in which said product is a necessarysubstrate.

The present inventors have determined that the advantages of performinga multi-step metabolic pathway in a BMC (concentration of enzymaticactivity and reaction substrates) can in fact be achieved in the absenceof a BMC. Furthermore, the present inventors have demonstrated that incells expressing multiple recombinant proteins comprising an enzymaticdomain and a BMC targeting signal polypeptide, increased product yieldis achieved in cells lacking BMCs as compared to those comprising BMCs.This finding was very surprising, and counterintuitive, given that theconsensus in the field prior to the present invention was that productyields would be increased by linking enzymes to BMC-targeting sequenceswith the specific purpose of recruiting said enzymes into co-expressedBMCs.

Furthermore, as shown in the present Examples, the addition ofBMC-targeting sequences to most enzymes reduces the specific activity ofthe enzymes. In addition, aggregation is usually considered to have adetrimental effect on protein function, and is therefore consideredundesirable. Therefore, the present inventors' finding that despitedecreased enzyme activity as a result of fusing BMC-targeting signalpolypeptides to said enzymes, and despite the absence of the BMCs, anincrease in product yield is observed in the microorganisms of theinvention, was very surprising.

The present invention is particularly advantageous in multi-steppathways, since a greater number of steps requires a greater number ofenzymatic domains and a greater number of interactions between theproduct(s) of one step and the active site of a subsequent enzymaticdomain. In such systems requiring numerous complex interactions, thespatial concentration of enzymatic domains and reaction substrates isparticularly advantageous.

Preferably, the microorganism of the invention comprises at least four,five, six, seven, eight or nine of said different recombinant proteins.Alternatively viewed, preferably, the microorganism of the inventioncomprises one or more heterologous nucleic acid molecules togetherencoding at least four, five, six, seven, eight or nine of saiddifferent proteins. Throughout this application, disclosures relating toone or more polypeptides or proteins are to be considered as disclosuresrelating to one or more nucleic acid molecules encoding saidpolypeptides or proteins, and vice versa.

The enzymatic domains as referred to herein each catalyse a differentsubstrate to product conversion in the same metabolic pathway. A“metabolic pathway” is a series of substrate to product conversions,each of which is catalysed by an enzyme, wherein the product of oneenzyme acts as the substrate for the next enzyme. The enzymatic domainsas referred to herein each catalyse a different substrate to productconversion in the same metabolic pathway for the production of a productof interest.

The microorganisms of the invention, also termed herein “microbes”,“microbial host cells” or simply “host cells” or “cells”, may be anymicroorganism in which recombinant proteins can be expressed. By“microorganism” is meant any unicellular prokaryotic or eukaryoticorganism. Preferred microorganisms are bacteria, cyanobacteria,microalgae, filamentous fungi and yeasts. Most preferably, themicroorganism is a bacterium.

As explained in more detail below, the invention provides methods ofproducing a product of interest comprising growing the microorganism ofthe invention in a culture medium and under conditions wherein theproduct is produced and optionally recovering the product. Depending onthe product of interest, the product may be secreted by themicroorganism and recovered from the culture medium, or the product maybe sequestered by the microorganism, necessitating extraction therefrom.In either case, to maximise production of the product of interest, themicroorganisms are preferably tolerant to the product of interest.

In some embodiments, the microorganisms have the ability to utilizecarbohydrates. Optionally, the microorganisms of the invention arephotosynthetic, preferably photosynthetic bacteria.

The microorganisms of the present invention comprise at least threerecombinant proteins and in some embodiments the microorganisms arefurther genetically modified to remove the ability of the cell to formBMCs. The ability to genetically modify the microorganism is essentialfor the production of any recombinant microorganism. Thus, preferablythe microorganisms are competent. “Competence” is the ability of a cellto take up extracellular nucleic acid molecules from its environment.The competence may be naturally occurring or induced, i.e. artificialcompetence, in which the microorganisms in culture are treated to makethem transiently permeable to DNA.

Preferably, the microorganisms of the present invention have the abilityto grow to high cell densities. It will be within the competencies ofthe person of ordinary skill in the art to determine the optimal celldensity for a particular microorganism and pathway of interest.Preferably, the microorganisms are thermophilic. Preferably, themicroorganisms are able to grow under anaerobic conditions.Alternatively preferably, the microorganisms are able to grow underaerobic conditions.

The above characteristics of the microorganism of the invention can beconferred by mutagenesis and selection, genetic engineering, or can benatural.

Preferably, the microorganism is selected from the group consisting ofClostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia,Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus,Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter,Corynebacterium, Brevibacterium, Schizosaccharomyces, Kluyveromyces,Yarrowia, Pichia, Candida, Hansenula, or Saccharomyces.

Preferably, the microorganism is a bacterium, more preferably of thegenus Escherichia, most preferably E. coli. E. coli is well establishedas an industrial microorganism used in the production of a variety ofproducts (chemical compounds, amino acids, vitamins, recombinantproteins). The entire E. coli genome has also been sequenced, and thegenetic systems are highly developed.

The preferred yeast organism is Saccharomyces cerevisiae. This organismhas a long history of use in industrial processes and can be manipulatedby both classical microbiological and genetic engineering techniques. Itis well-characterized genetically; the entire genome of S. cerevisiaehas been sequenced. The organism grows to high cell densities.

Preferred microalgae for use in the present invention include Chlorellaand Prototheca.

As mentioned above, the microorganism of the invention is essentiallyfree of BMCs, i.e. expresses essentially no BMCs. Preferably, themicroorganism of the invention is free of BMCs, i.e. does not expressBMCs. Preferably, the microorganism of the invention does not naturallyexpress BMCs, i.e. does not naturally comprise the genes necessary forthe expression of BMCs. By “naturally” in this context is meant“natively”, i.e. prior to any modification according to the invention.In such microorganisms, no genetic modification is required to preventthe microorganism from expressing BMCs. Throughout the application, theterms “express” and “expresses” are interchangeable with the term“having the ability to express”, i.e. comprising the genes necessary forexpression.

Alternatively, preferably, the microorganism of the invention naturallyexpresses BMCs but has been modified to reduce essentially all,preferably all, of the ability to express BMCs. In other words, themicroorganism of the invention is preferably of a species or strain thatnatively expresses BMCs but has been modified to reduce essentially all,preferably all, of the cell's ability to express BMCs. Suitablemodifications to achieve this reduction are discussed in more detailelsewhere herein.

In nature, microorganisms that naturally express BMCs typically do soonly under certain conditions, namely in the presence of inducermolecules, which for any given BMC is the substrate for the pathway thatcomprises steps catalysed by enzymes located within the BMCs. Forinstance, Pdu BMCs are only expressed by microorganisms comprising thenecessary genes when said microorganisms are exposed to 1,2-propanediol.Similarly, Eut BMCs are only expressed by microorganisms comprising thenecessary genes when said microorganisms are exposed to ethanolamine.

Thus, in an alternative embodiment, the microorganism of the inventionis of a species or strain that naturally expresses BMCs, i.e. thatcomprises the genes necessary for the expression of BMCs, and whereinexpression of said BMCs is inducible by the presence of one or moreinducer molecules, but wherein said microorganism is in an environment,e.g. a culture medium, that does not permit expression of BMCs. In otherwords, preferably the genetically modified microorganism of theinvention is present in a culture medium in which the level of saidinducer molecule(s) is too low to induce the expression of said BMCs.Preferably the culture medium lacks said inducer molecule(s).

If the microorganism naturally expresses Pdu BMCs, then said molecule is1,2-propanediol. If the microorganism expresses Eut BMCs, then saidmolecule is ethanolamine. Expression of other BMCs is known to beinduced by the presence of choline, fucose or rhamnose.

Thus, the present invention provides a microorganism of the inventionpresent in a culture medium in which the level of said inducermolecule(s) is too low to induce the expression of said BMCs. Preferablythe culture medium lacks said inducer molecule(s). Alternatively viewed,the present invention provides a culture medium comprising amicroorganism of the invention that naturally expresses BMCs, i.e. thatcomprises the genes necessary for the expression of BMCs, wherein insaid culture medium the level of said inducer molecule(s) is too low toinduce the expression of said BMCs. Preferably the culture medium lackssaid inducer molecule(s). Preferably, the culture media lacks one ormore, preferably all of propanediol, ethanolamine, choline, fucose andrhamnose.

Microorganisms that possess the ability to produce BMCs, i.e. naturallyexpress, i.e. natively express one or more of Pdu, Eut and carboxysomeBMCs, or less common BMCs, are known in the art, for instance from Axenet al., (2014) PLOS Computational Biology 10(10):e1003898, US2012/0210459, and Jorda J, et al., (2013) Protein Science: A Publicationof the Protein Society. 22(2):179-195. Dataset S1 of Axen et al.comprehensively lists bacterial strains that possess the ability toproduce BMCs, i.e. naturally comprise the genes necessary for productionof BMCs. The skilled person would be able to determine whether or not aparticular microorganism possesses the ability to produce BMCs, i.e.comprises the genes necessary for the expression of BMCs.

The microorganisms of the present invention are genetically modified toincrease the yield of the product of interest. As used herein, the term“wild type microorganism” or “wild type cell” encompasses the typical,i.e. most prevalent microorganism of a species or strain as it occurs innature. Existing strains of a particular species are not necessarily“wild type” strains, however, existing strains lack the modifications ofthe invention described herein. The term “wild type” is used herein asshorthand to refer to microorganisms of the same strain as themicroorganism of the invention but lacking the genetic modifications ofthe invention as described herein, even though such microorganisms maynot be the most prevalent strain. This is how the term is typically usedin the field.

The microorganisms of the invention are recombinant microorganisms, i.e.they comprise one or more recombinant nucleic acid molecules. Cellsand/or microorganisms may be genetically modified by genetic engineeringtechniques (e.g., recombinant technology), classical microbiologicaltechniques, or a combination of such techniques. Such techniques aregenerally disclosed, for example, in Sambrook et al., 1989, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Labs Press.

The genetically modified microorganisms of the invention can include amicroorganism in which nucleic acid molecules have been inserted,deleted or modified (i.e., mutated; e.g., by insertion, deletion,substitution, and/or inversion of nucleotides), in such a manner thatsuch modifications provide the desired effect of increased yields of theproduct of interest within the microorganism or in the culture medium.

As used herein, genetic modifications which result in a decrease in geneexpression, in the function of the gene, or in the function of the geneproduct (i.e. the protein encoded by the gene) can be referred to asinactivation (complete or partial), deletion, interruption, blockage ordown-regulation of a gene. They can be referred to as null mutations orloss of function mutations. For example, a genetic modification in agene which results in a decrease in the function of the protein encodedby such gene, can be the result of a complete deletion of the gene(i.e., the gene does not exist, and therefore the protein is notproduced), a mutation in the gene which results in incomplete or notranslation of the protein (e.g., the protein is not expressed), or amutation in the gene which decreases or abolishes the natural functionof the protein (e.g., a protein is expressed which has decreased or noenzymatic activity). Genetic modifications which result in an increasein gene expression or function can be referred to as amplification,overproduction, overexpression, activation, enhancement, addition, orup-regulation of a gene. The terms “gene expression” and “proteinexpression” are used interchangeably herein. Methods and types ofmutation are well-known in the art and any suitable method or type canbe present in the microorganisms of the present invention. Mutationsinclude, for instance, missense mutations, nonsense mutations,insertions, deletions, duplications, frameshift mutations and repeatexpansions, and any combination thereof.

Addition of recombinant genes to increase gene expression can includemaintaining the recombinant gene(s) on replicating plasmids orintegrating the recombinant gene(s) into the genome of the productionorganism. Furthermore, increasing the expression of desired recombinantgenes can include operatively linking the recombinant gene(s) to nativeor heterologous transcriptional control elements.

The microorganisms of the invention comprise one or more heterologousnucleic acid molecules together encoding at least three differentproteins (that are by definition recombinant proteins). Alternativelyviewed, the microorganisms of the present invention comprise at leastthree different recombinant proteins. Preferably, each of said proteinsis encoded by a different recombinant nucleic acid molecule.Alternatively viewed, preferably each of said heterologous nucleic acidmolecules encodes only one of said proteins. Alternatively, two, three,or more of said proteins are encoded by the same recombinant nucleicacid molecule. Preferably, the microorganism of the invention comprises3, 4, 5, 6, 7, 8 or 9 recombinant proteins as defined herein.

As used herein, the term “protein” means a polymer of amino acidresidues. The terms “polypeptide” and “protein” are used interchangeablyherein. The recombinant proteins of the invention each comprise a regionhaving enzymatic activity and a BMC-targeting signal polypeptide. A mereoligopeptide comprising 2 (a dipeptide), 3 (a tripeptide) or up to about25 amino acids is not sufficiently long to comprise a region withenzymatic activity and a BMC-targeting sequence. The proteins of theinvention are preferably each a polypeptide comprising at least 75 aminoacids, more preferably at least 100 amino acids, still more preferablyat least 120 amino acids.

Both full length proteins and fragments thereof are contemplated by theterm “protein” as used herein. “Fragments” in the context of the presentinvention are functional fragments, i.e. a fragment comprises the sameenzymatic activity as the full length protein of which it is a fragment.The term “protein” also includes post-expression modifications to theprotein, including, but not limited to, glycosylation, acetylation andphosphorylation. The term “protein” also applies to amino acid polymersin which one or more amino acid residue is an artificial chemicalmimetic of a corresponding naturally occurring amino acid, as well as tonaturally occurring amino acid polymers and non-naturally occurringamino acid polymers. Amino acid polymers may comprise entirely L-aminoacids, entirely D-amino acids, or mixture of L- and D-amino acids.

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein to refer to deoxyribonucleotides or ribonucleotides and polymersthereof in either single- or double-stranded form. The term encompassesnucleic acids containing known nucleotide analogs or modified backboneresidues or linkages, which are synthetic, naturally occurring, andnon-naturally occurring, which have similar binding properties as thereference nucleic acid, and which are metabolized in a manner similar tothe reference nucleotides. Examples of such analogs include, withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiral-methyl phosphonates, 2-O-methyl ribonucleotides,polypeptide-nucleic acids (PNAs). Unless otherwise indicated, aparticular nucleic acid sequence also encompasses “conservativelymodified variants” thereof (e.g., degenerate codon substitutions) andcomplementary sequences, as well as the sequence explicitly indicated.

As used herein, the term “heterologous” when applied to a nucleic acidmolecule or protein means a nucleic acid molecule or protein that is notnaturally, i.e. natively, present or encoded in the genome of thatstrain of microorganism. Thus, heterologous nucleic acid molecules arethose that are introduced into the microorganism by recombinanttechniques. The terms “non-native” and “heterologous” are usedinterchangeably. In the context of the present invention, the terms“heterologous” and “recombinant” are used interchangeably. As usedherein, the term “native” when applied to a nucleic acid molecule orprotein means a nucleic acid molecule or protein that is present orencoded in the genome of that strain of microorganism.

The microorganism of the present invention comprises one or moreheterologous nucleic acid molecules that encode at least three proteins,each protein comprising an enzymatic domain and a bacterialmicrocompartment-targeting signal polypeptide. In such a heterologousnucleic acid molecule, the coding sequence for the enzymatic domain maybe native to the microorganism and the coding sequence for theBMC-targeting signal polypeptide may be non-native, or vice versa.Alternatively, both the coding sequence for the enzymatic domain and thecoding sequence for the BMC-targeting signal polypeptide may benon-native to the microorganism, and may be from the same or differentnon-native sources. Alternatively, the coding sequence for the enzymaticdomain and the coding sequence for the BMC-targeting signal polypeptidemay both be native to the microorganism but the nucleic acid moleculecomprises one or more additional sequences that are non-native to themicroorganism. These additional sequences may encode for other sequenceswithin the encoded protein such as linker sequences between theenzymatic domain and the BMC-targeting signal polypeptide, or they maybe regulatory sequences such as promoters. Alternatively, the codingsequence for the enzymatic domain and the coding sequence for theBMC-targeting signal polypeptide may be native to the microorganism butare not found natively in the same nucleic acid molecule, such thatoverall the nucleic acid molecule of the invention is heterologous tothe microorganism of the invention.

The heterologous nucleic acid molecules of the present invention arerecombinant nucleic acid molecules. Recombinant nucleic acid molecules,also known as “chimeric nucleic acid molecules” are nucleic acidmolecules formed by laboratory methods of genetic recombination (such asmolecular cloning) to combine nucleic acid sequences from two or moresources.

A “recombinant protein” is a protein that is encoded by a recombinantnucleic acid molecule, preferably by recombinant DNA (also termed“chimeric DNA”). A recombinant protein is encoded by a recombinant gene,i.e. by a chimeric gene, specifically by the coding region(s) of thechimeric gene. Thus, the recombinant nucleic acid molecules of thepresent invention comprise at least three different chimeric genesencoding the at least three different proteins defined herein.Recombinant protein expression is the expression of proteins within acell from recombinant DNA. The at least three different proteins thatthe microorganisms of the invention comprise are recombinant proteins,and the terms “protein” and “recombinant protein” are usedinterchangeably in this context throughout the application.

“Chimeric gene” refers to any gene that is not a native gene. A chimericgene as used herein comprises regulatory and coding sequences that arenot found together in nature and/or a coding sequence comprising two ormore sequence regions not found together in nature. Accordingly, achimeric gene may comprise regulatory sequences and coding sequencesthat are derived from different sources, or regulatory sequences andcoding sequences derived from the same source, but arranged in a mannerdifferent than that found in nature. A “foreign gene”, “non-native” or“heterologous gene” refers to a gene not normally found in the hostorganism, but that is introduced into the host organism by genetransfer. Foreign genes can comprise genes native to one organisminserted into a different, i.e. non-native, organism, or they cancomprise chimeric genes. A “transgene” is a gene that has beenintroduced into the genome by a transformation procedure.

As explained in more detail below, the enzymatic domain (also termed“the region with enzymatic activity” herein) and the BMC-targetingsignal polypeptide of a recombinant protein of the invention preferablyoriginate from different sources, i.e. from different organisms or fromdifferent proteins within a single organism. In these embodiments, thecoding sequence within the recombinant nucleic acid molecule is achimeric sequence.

In other embodiments, the enzymatic domain and the BMC-targeting signalpolypeptide of a recombinant protein of the invention originate from thesame source, preferably from a microorganism that naturally expresses aprotein comprising said enzymatic domain and said BMC-targeting signalpolypeptide. Optionally, this microorganism is a wild type organism ofthe same species as the microorganism of the invention, however,typically this is not the case. In these embodiments, the codingsequence within the recombinant nucleic acid molecule is not a chimericsequence, however, the recombinant nucleic acid molecule as a whole is achimeric sequence due to the coding sequence being operably linked toone or more regulatory sequences, wherein the coding sequence and theone or more regulatory sequences are from different sources. If two ormore regulatory sequences are present, they are preferably fromdifferent sources from each other. Preferably, the regulatorysequence(s) is/are non-native to the microorganism of the invention.

As used herein, the term “gene” refers to a nucleic acid that is capableof being expressed as a specific polypeptide, optionally includingregulatory sequences preceding (5′ non-coding sequences) and following(3′ non-coding sequences) the coding sequence. “Native gene” or “wildtype gene” refers to a gene as found in nature with its own regulatorysequences. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism.

As used herein, the term “coding sequence” refers to a nucleic acidsequence that codes for a specific amino acid sequence. “Regulatorysequences” refer to nucleotide sequences located upstream (5′ non-codingsequences), within, or downstream (3′ non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence. Regulatorysequences may include promoters, translation leader sequences, introns,polyadenylation recognition sequences, RNA processing site, effectorbinding site and stem-loop structure.

The term “promoter” refers to a nucleic acid sequence capable ofcontrolling the expression of a coding sequence or functional RNA. Ingeneral, a coding sequence is located 3′ to a promoter sequence.Promoters may be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, or even comprise synthetic DNA segments. It is understood bythose skilled in the art that different promoters may direct theexpression of a gene in different tissues or cell types, or at differentstages of development, or in response to different environmental orphysiological conditions. Promoters which cause a gene to be expressedin most cell types at most times are commonly referred to as“constitutive promoters”. It is further recognized that since in mostcases the exact boundaries of regulatory sequences have not beencompletely defined, DNA fragments of different lengths may haveidentical promoter activity.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. The terms “gene expression” and“protein expression” are used interchangeably herein.

The microorganism of the invention is prepared by the transformation ofa microorganism with the one or more heterologous nucleic acid moleculestogether encoding at least three different proteins, each proteincomprising an enzymatic domain and a bacterialmicrocompartment-targeting signal polypeptide, wherein said enzymaticdomains each catalyse a different substrate to product conversion in thesame metabolic pathway.

As used herein the term “transformation” refers to the transfer of anucleic acid fragment into a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

The one or more heterologous nucleic acid molecules of the inventionthat encode the at least three different proteins are each preferablycomprised within a plasmid, vector or cassette. The terms “plasmid”,“vector” and “cassette” refer to an extra chromosomal element oftencarrying genes which are not part of the central metabolism of the cell,and usually in the form of circular double-stranded DNA fragments. Suchelements may be autonomously replicating sequences, genome integratingsequences, phage or nucleotide sequences, linear or circular, of asingle- or double-stranded DNA or RNA, derived from any source, in whicha number of nucleotide sequences have been joined or recombined into aunique construction which is capable of introducing a promoter fragmentand DNA sequence for a selected gene product along with appropriate 3′untranslated sequence into a cell. “Transformation cassette” refers to aspecific vector containing a foreign gene and having elements inaddition to the foreign gene that facilitates transformation of aparticular host cell.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter. The one ormore heterologous nucleic acids of the invention are typically presentin an expression vector, preferably a vector comprising a strongpromoter.

Preferably, the at least three recombinant proteins are eachover-expressed in the microorganism of the invention. As referred toherein, “over-expressed” means that expression of the gene encoding theprotein is increased as compared to, i.e. relative to, the level ofexpression in a control microorganism, i.e. in a microorganism in thesame strain which lacks said one or more heterologous nucleic acidmolecules. The skilled person would appreciate that the comparison mustbe made between the level of expression of a protein in themicroorganism of the invention and the level of expression of the sameprotein (i.e. a protein having the same amino acid sequence) occurringin the control microorganism.

A “control microorganism” is a microorganism of the same strain as themicroorganism of the invention which has not been modified according tothe invention. In this context of over-expression, a “control organism”has not been modified to over-express the gene in question. A controlorganism for instance may have been transformed with an “empty” vectoror a vector with a control sequence. Preferably, the controlmicroorganism is one that does not comprise the one or more heterologousnucleic acid molecules of the invention that encode a protein comprisingan enzymatic domain and a bacterial microcompartment-targeting signalpolypeptide. The skilled person will readily be able to determine theappropriate control with which to make the comparison of expressionlevels.

Gene expression is to be considered in terms of the amount of proteinproduct produced, which may be determined by any convenient method knownin the art. The terms “gene expression” and “protein expression” areused interchangeably herein. Methods for the direct and indirectdetermination of protein expression levels are well-known in the art andany such technique could be used by the skilled person in this regard.For example, expression can be determined by measuring protein activity.Alternatively, the amount of protein produced can be measured todetermine the level of expression, for example by Western Blotting orother antibody detection systems, or indeed by any method of assessingor quantifying protein. The assay may be an in vivo or in vitro assay.

In some embodiments, the microorganism of the invention comprises arecombinant protein that is not also expressed by the controlmicroorganism, in which case the level of expression of said protein inthe control microorganism is zero. This is the situation, for instance,when the control microorganism is the same strain as the microorganismof the invention and wherein said strain does not naturally express saidprotein. In other embodiments, the microorganism of the inventioncomprises a recombinant protein that is also expressed by the controlmicroorganism, in which case the level of expression of the protein inthe microorganism of the invention nevertheless exceeds that in thecontrol microorganism.

Thus, the microorganisms of the present invention preferably comprise atleast three different recombinant proteins that are each over-expressedrelative to the expression level of said protein in a controlmicroorganism as defined above.

Preferably, the one or more heterologous nucleic acid molecules of thepresent invention together comprise at least three different chimericgenes, each comprising a coding sequence that encodes a differentprotein comprising an enzymatic domain and a BMC-targeting signalpolypeptide as defined anywhere herein operably linked to one or moreregulatory elements for the over-expression of said protein. Preferably,said regulatory element is a promoter, more preferably a strongpromoter. Thus, preferably each of the heterologous nucleic acidmolecules comprises a region encoding one or more of said proteinsoperably linked to a strong promoter.

Alternatively viewed, the present invention provides a geneticallymodified microorganism comprising one or more heterologous nucleic acidmolecules together encoding at least three different proteins, eachprotein comprising an enzymatic domain and a bacterialmicrocompartment-targeting signal polypeptide, wherein said enzymaticdomains each catalyse a different substrate to product conversion in thesame metabolic pathway, wherein each of said proteins is over-expressed,and wherein said microorganism is essentially free of bacterialmicrocompartments.

Alternatively viewed, the present invention provides a geneticallymodified microorganism comprising at least three different proteins,each protein comprising an enzymatic domain and a bacterialmicrocompartment-targeting signal polypeptide, wherein said enzymaticdomains each catalyse a different substrate to product conversion in thesame metabolic pathway, wherein each of said proteins is over-expressedand wherein said microorganism is essentially free of bacterialmicrocompartments.

Preferably, “over-expressed” or “over-expression” means that the levelof expression is at least 10%, preferably at least 20%, even morepreferably at least 50%, yet more preferably at least 75%, still morepreferably at least 90%, and most preferably at least 100% greater inthe microorganism of the invention as compared to the level ofexpression in the control microorganism as defined above. Alternatively,expression may be 2-, 3- or 4-fold or more higher in the microorganismof the invention as compared to the level in the control microorganismdefined above.

Over-expression of a protein of interest can be achieved by anytechnique known in the art, and such techniques would be well known toone of ordinary skill in the art. According to the present invention“overexpressing” may mean simply that an additional gene is expressed inthe microorganism beyond the native gene endogenously present in thatmicroorganism but is not limited to such a mechanism. It may includeexpressing a gene in a microorganism which does not naturally containsuch a gene.

Preferably, the microorganism of the invention comprises aggregatescomprising said at least three different proteins.

Over-expression is preferably achieved by introducing into themicroorganism a recombinant nucleic acid molecule encoding the protein,for example expressed from a stronger or unregulated promoter relativeto the gene in the control microorganism, and/or by introducing multiplecopies of a protein-encoding nucleic acid molecule.

Preferably, the introduced nucleic acid molecule is modified as comparedto a naturally occurring gene encoding the same protein to render itrelieved of transcriptional repression, e.g. by mutating or deletingrecognition elements for transcriptional repressors or by usingexpression control elements (e.g. promoters) which are not subject totranscriptional regulation by the transcriptional regulator(s) whichnormally control expression of the gene. The endogenous gene mayalternatively or additionally be modified in this way, or by addition ofa stronger promoter. Thus, mutagenesis (including both random andtargeted) may for example be used to mutate the endogenous control orregulatory elements so as to increase expression of the endogenous gene(e.g. increase transcription and/or translation). Alternatively, theorganism may be engineered to introduce additional or alternativeregulatory elements.

In a particular embodiment, a gene may be expressed from a non-native orheterologous promoter (that is a promoter which is heterologous to theencoding gene, i.e. is not the native gene promoter) and particularly astrong, non-native or heterologous promoter. Thus, in this embodimentthe gene is not used with its native promoter. A gene may be introducedwhich is under the control of a non-native promoter. As referred toherein, a strong promoter is one which expresses a gene at a high level,or at least at a higher level than effected by its native promoter. Theterm “strong promoter” is a term well known and widely used in the artand many strong promoters are known in the art, or can be identified byroutine experimentation. The use of a non-native promoter mayadvantageously have the effect of relieving the gene of transcriptionalrepression, as at least some of any repressive elements will be locatedin the native promoter region. By replacing the native promoter with anon-native promoter devoid of repressive elements responsive to theeffects of pathway products, the gene will be at least partly relievedof transcriptional repression.

Suitable promoters and expression vectors comprising such promoters forachieving over-expression of a protein of interest are well-known in theart, and the one or more nucleic acid molecules of the invention maycomprise any such promoter or may be comprised within any suchexpression vector to achieve over-expression of the at least threeproteins of the invention. Examples include the E. coli expressionvector pGEX in which protein expression is under the control of the tacpromoter, and the pET series of vectors which uses a T7 promoter.

As used herein, the term “enzymatic domain” means the region of theprotein that performs and is necessary for the catalysis of a substrateto product conversion. The terms “region having enzymatic activity” and“enzymatic domain” are used interchangeably herein. The enzymatic domainmay be a complete enzyme or a function fragment thereof, i.e. a fragmentthat catalyses the same substrate to product conversion as the completeenzyme. The enzymatic domain comprises a catalytic domain, i.e. anactive site, and any other amino acids necessary for the domain to havea conformation that permits said active site to be functional, i.e. toperform catalysis in the presence of the relevant substrates.

In one or more of the recombinant proteins of the invention, theBMC-targeting signal polypeptide may be fully comprised within or may bepartially comprised within, i.e. may overlap with, the enzymatic domain.Alternatively viewed, the enzymatic domain may comprise all or part ofthe BMC-targeting signal polypeptide. In such embodiments, the presenceof part or all of the BMC-targeting signal polypeptide is necessary forthe function of the enzymatic domain. In such embodiments, the sequencesof the BMC-targeting signal polypeptide and the enzymatic domainoverlap, at least partially.

Preferably, however, the enzymatic domain and the BMC-targeting signalpolypeptide are distinct domains. By distinct in this context is meantthat the sequences of the BMC-targeting signal polypeptide and theenzymatic domain do not overlap, i.e. the domains are structurallydistinct, i.e. have distinct amino acid sequences. In such embodiments,the BMC-targeting signal polypeptide is not comprised fully or partiallywithin the enzymatic domain, i.e. the enzymatic domain does not compriseall or part of the BMC-targeting signal polypeptide. In theseembodiments, the sequence of the enzymatic domain and the BMC-targetingsignal polypeptide may be directly adjacent, i.e. the C-terminal aminoacid of the BMC-targeting signal polypeptide may be adjacent in sequenceto the N-terminal amino acid of the enzymatic domain, or the C-terminalamino acid of the enzymatic domain may be adjacent in sequence to theN-terminal amino acid of the BMC-targeting signal polypeptide.

Optionally, the enzymatic domain and the BMC-targeting signalpolypeptide are linked by an amino acid linker. The amino acid linkermay comprise any number of amino acids. Preferably, the amino acidlinker is 1 to 60 amino acids in length, more preferably 2 to 40 aminoacids in length, most preferably 4 to 30 amino acids in length. Theamino acid linker sequence may itself be a protein or polypeptide withstable secondary structure, i.e. a rigid linker, such as an alphahelical or beta sheet structure, and optionally with tertiary structure.Preferably, however, the amino acid linker sequence lacks stablesecondary structure. Instead, it is preferably a random coil. A randomcoil is a sequence of amino acids with a conformation in which the aminoacids are oriented randomly while still being bonded to adjacent aminoacids.

Optionally, the linker comprises one or more sequences that assist withprotein purification. In this regard, preferably, the amino acid linkercomprises a sequence of 2 to 15, more preferably 2 to 12 or 3 to 12,still more preferably 2 to 6 or 3 to 6 consecutive histidine residues.Optionally, the linker comprises one or more cleavage sites.

Preferably, the BMC-targeting signal polypeptide is N-terminal to theenzymatic domain, optionally separated by a linker sequence as describedabove.

Preferably, the BMC-targeting signal peptide is located at theN-terminus of the protein. However, it is possible for the protein tocomprise amino acids N-terminal to the BMC-targeting signal peptide.

Alternatively preferably, the BMC-targeting signal polypeptide isC-terminal to the enzymatic domain, optionally separated by a linkersequence as described above.

Preferably, the BMC-targeting signal peptide is located at theC-terminus of the protein. However, it is possible for the protein tocomprise amino acids C-terminal to the BMC-targeting signal peptide.

As used herein, the term “distinct” also requires the domains to befunctionally distinct. “Functionally distinct” as used herein means thatthe function of one of the domains does not require the presence of theother domain. In the context of the present invention, the two domainsare the enzymatic domain and the BMC-targeting signal polypeptide. Thus,in these embodiments, the presence of the BMC-targeting signalpolypeptide is not necessary for the function of the enzymatic domain,and vice versa. In such embodiments, cleavage of the BMC-targetingsignal polypeptide does not remove the catalytic ability of theenzymatic domain. If the BMC-targeting signal polypeptide was necessaryfor the catalytic function of the enzymatic domain, then the domainswould not be considered “distinct” as the term is used herein, since theenzymatic domain is the region of the protein that performs and isnecessary for the catalysis of a substrate to product conversion and sowould include the BMC-targeting signal polypeptide.

Preferably one or more, more preferably all, of the recombinant proteinsof the invention are “bipartite proteins”. A bipartite protein is aprotein with at least two functionally distinct domains. A bipartiteprotein of the present invention may comprise more than two functionallydistinct domains but as a minimum it must contain an enzymatic domainand a BMC-targeting signal polypeptide that are functionally distinct.

Optionally, one or more of the recombinant proteins is “native” to themicroorganism of the invention. By “native” is meant that themicroorganism of the invention is of a strain that naturally expresses aprotein with the same amino acid sequence. A recombinant protein is bydefinition not endogenously expressed by a microorganism because it isexpressed from a recombinant nucleic acid molecule, however, the aminoacid sequence of a recombinant protein may be identical to a proteinexpressed endogenously by the microorganism, in which case therecombinant protein is said to be native to the microorganism.

One or more of the recombinant proteins may comprise an enzymatic domainthat is native and a BMC-targeting signal polypeptide that is non-nativeto the microorganism of the invention. Alternatively, one or more of therecombinant proteins may comprise a BMC-targeting signal polypeptidethat is native and an enzymatic domain that is non-native to themicroorganism of the invention. In these embodiments, the non-nativecomponents of the one or more recombinant proteins are preferably nativeto a different species of microorganism, i.e. a microorganism of aspecies other than the species of the microorganism of the invention.Alternatively, the non-native components are artificial, i.e. have asequence not found in nature.

Optionally, the enzymatic domain and the BMC-targeting signalpolypeptide of a recombinant protein are both native to themicroorganism of the invention but are expressed as parts of differentnative proteins. In this latter embodiment, the recombinant protein is,overall, non-native to the microorganism of the invention because thestrain does not naturally express a protein with the same overallsequence.

If present, amino acid linker(s) between the enzymatic domain and theBMC-targeting signal polypeptide may be native or non-native to themicroorganism of the invention. Similarly, any other region of therecombinant protein may be native or non-native to the microorganism ofthe invention.

Preferably, one or more, more preferably all of the recombinant proteinsare non-native to the microorganism of the invention. By “non-native” ismeant “heterologous”, i.e. that the microorganism is of a strain thatdoes not naturally express a protein with the same sequence. Theequivalent definition of “non-native” applies in the context of thenon-native enzymatic domains, non-native BMC-targeting signalpolypeptides and non-native nucleic acid molecules disclosed herein.

Non-native proteins, parts/regions of proteins, polypeptides, domainsand nucleic acid molecules as referred to herein may each occur innature, i.e. within a different organism from that of the invention, ormay be artificial, i.e. not found anywhere in nature.

In a preferred embodiment, one or more of the recombinant proteins isnon-native to the microorganism of the invention but is native to, i.e.expressed naturally by, another microorganism. Many bipartite proteinsthat comprise an enzymatic domain and a BMC-targeting signal sequenceare known in the art. Examples of such enzymes include the dioldehydratase (PduDE), the propionaldehyde dehydrogenase (PduP), thephosphotransacylase (PduL) and the 1-propanol dehydrogenase (PduQ) ofmicroorganisms naturally expressing Pdu BMCs, the aldehyde dehydrogenase(EutE) and the ethanolamine deaminase (EutC) of microorganisms naturallyexpressing Eut BMCs, and the gamma-carbonic anhydrase (CcmM) ofmicroorganims naturally expressing carboxysomes.

If the recombinant protein comprises an enzymatic domain and aBMC-targeting polypeptide, both of which are non-native to themicroorganism of the invention, then optionally said enzymatic domainand said BMC-targeting domain are expressed within different proteins bya single other microorganism species, or within proteins expressed bytwo different other microorganism species. Alternatively, one or more ofsaid domains and polypeptides may be artificial.

Optionally, the enzymatic domain and/or the BMC-targeting sequence, anyportion of the recombinant protein, or the entire recombinant protein isartificial, i.e. has an amino acid sequence not found in nature.

Preferably, one or more, more preferably all, of the recombinantproteins of the present invention are fusion proteins. As used herein, afusion protein is a single protein having at least two domains that arenot present in the same protein in nature. Naturally occurring proteinsare thus not “fusion proteins” as the term is used herein. In thepreferred fusion proteins of the present invention, the enzymatic domainand the BMC-targeting signal polypeptide are not present in the sameprotein in nature. Optionally, the enzymatic domain and theBMC-targeting signal polypeptide of a fusion protein of the inventionare expressed within different naturally occurring proteins in the sameorganism, which is optionally the wild type organism of the same speciesas the microorganism of the invention, but preferably an organism of adifferent species. Alternatively, the two domains are expressednaturally within different organisms. Fusion proteins are preferredbipartite proteins of the invention. The fusion proteins of theinvention may also comprise or consist of artificial sequences.

As used herein, a “fusion protein construct” is a nucleic acid constructthat is composed of different genes or portions thereof in operablelinkage. The components include a nucleic acid molecule encoding atleast an enzymatic domain as defined herein and a nucleic acid moleculeencoding at least a BMC targeting signal polypeptide as defined herein.

Thus, typically the microorganisms of the invention comprise at leastthree different recombinant proteins that are not expressed naturally bythat strain of microorganism. In some embodiments, however, one or moreof the recombinant proteins is identical in amino acid sequence to aprotein expressed naturally by that strain of microorganism.

The disclosure herein relating to proteins and parts thereof appliesmutatis mutandis to nucleic acid molecules encoding said proteins andparts thereof.

The microorganisms of the invention comprise at least three differentrecombinant proteins that each comprise an enzymatic domain, wherein theenzymatic domains each catalyse a different substrate to productconversion in the same metabolic pathway. In the presence of suitablesubstrates, the microorganism of the invention produces the product ofthe metabolic pathway.

The product produced by the microorganism of the present invention, i.e.the product of interest, may be any product of a metabolic pathway thatcomprises at least three enzyme catalysed reactions. Preferably, themetabolic pathway for the product of interest comprises the formation ofone or more toxic intermediates, and preferably one or more of therecombinant proteins of the invention catalyses a step in which saidtoxic intermediate is a substrate or product. Preferred products areorganic compounds, preferably alcohols. Preferred alcohols are C₁ to C₁₂alcohols, more preferably C₂ to C₆ alcohols. Preferred alcohols areselected from the group consisting of ethanol, 1-propanol, 2-propanol,1-butanol, 2-butanol, isobutanol, tert-butanol, 1,2-propanediol,1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol and2,3-butanediol.

If the product of interest is 1,2-propanediol, then the enzymaticdomains each catalyse a different substrate to product conversionselected from:

-   -   i) glycerol to dihydroxyacetone    -   ii) dihydroxyacetone to dihydroxyacetone phosphate    -   iii) dihydroxyacetone phosphate to methylglyoxal    -   iv) methylglyoxal to lactaldehyde    -   v) lactaldehyde to 1,2-propanediol

Preferably, the microorganism comprises at least three differentrecombinant proteins comprising enzymatic domains that each catalyse adifferent substrate to product conversion selected from those listedabove. More preferably, the microorganism comprises four recombinantproteins comprising enzymatic domains that each catalyse a differentsubstrate to product conversion selected from those listed above.

Preferably said enzymatic domains are present in the form of completeenzymes. Preferably, the enzyme that catalyses the conversion ofglycerol to dihydroxyacetone is of the class EC 1.1.1.6, and ispreferably glycerol dehydrogenase. Preferably the enzyme that catalysesthe conversion of dihydroxyacetone to dihydroxyacetone phosphate is ofthe class EC 2.7.1.29, and is preferably dihydroxyacetone kinase.Preferably, the enzyme that catalyses the conversion of dihydroxyacetonephosphate to methylglyoxal is of the class EC 4.2.3.3, and is preferablymethylgloxal synthase. Preferably, the enzyme that catalyses theconversion of methylglyoxal to lactaldehyde is of the class EC 1.1.1.6,and is preferably glycerol dehydrogenase. Preferably, the enzyme thatcatalyses the conversion of lactaldehyde to 1,2-propanediol is of theclass EC 1.1.1.77, and is preferably 1,2-propanediol oxidoreductase.

Thus, preferably, the microorganism of the invention comprises at leastthree different recombinant proteins, each protein comprising adifferent enzyme and a bacterial microcompartment-targeting signalpolypeptide, wherein each of said enzymes is selected from those listedabove, and wherein said microorganism is essentially free of bacterialmicrocompartments. Such organisms produce 1,2-propanediol in thepresence of a suitable substrate, such as glycerol. Preferably, themicroorganism comprises all four of the above enzymes.

Each of the at least three proteins of the invention comprises aBMC-targeting signal polypeptide. A BMC-targeting signal polypeptide isa polypeptide that, when present within a protein, mediates theencapsulation of said protein within a BMC, if present. Suchpolypeptides are known in the art and the proteins of the invention cancomprise any such polypeptide. Two, three or more of the at least threeproteins of the invention may comprise the same BMC-targeting signalpolypeptide. Alternatively, each protein may comprise a differentBMC-targeting signal polypeptide.

In nature, BMC proteins are typically expressed from the same operon asthe proteins, e.g. enzymes, that comprise BMC-targeting polypeptides andthat localise within the BMCs. Thus, in nature, BMC-targeting signalpolypeptides tend to localise within a particular type of BMC, i.e. aredirected to a particular type of BMC. For example, in the Pdu operon,the Pdu BMC capsule proteins are expressed from the same operon as thePdu enzymes, and the BMC-targeting signal sequences within said Pduenzymes are localise to, i.e. are directed to, the Pdu BMCs. The terms“BMC-targeting signal polypeptide” and “BMC-targeting signal sequence”are used interchangeable herein.

US 2013/0133102 discloses known BMC-targeting signal polypeptides andthe types of BMCs to which they are directed. The BMC-targeting signalpolypeptides disclosed in this document may be used as BMC-targetingsignal polypeptides according to the present invention andUS2013/0133102 is incorporated herein by reference. Optionally, theBMC-targeting signal polypeptide of the present invention is aBMC-targeting polypeptide disclosed as such in US 2013/0133102.

The BMC-targeting signal polypeptides of the invention each preferablycomprise a region with alpha-helical conformation. Preferably theBMC-targeting signal polypeptides of the invention each comprise anamphipathic alpha helix. Preferably, the BMC-targeting signalpolypeptide is adjacent to an N-terminal and/or a C-terminal regionwithout stable secondary structure, i.e. a random coil. Methods ofpredicting the secondary structure of a given amino acid sequence arewell-known in the art, as are methods of designing a synthetic aminoacid sequence with a desired secondary structure.

The term “amphipathic alpha helix” or “amphipathic α-helix” refers to apolypeptide sequence that can adopt a secondary structure that ishelical with one surface, i.e., face, being polar and the other surfacebeing a nonpolar face. Typically, the polar face comprises primarilypolar and/or charged amino acids and the non-polar face comprisesprimarily hydrophobic amino acids. Methods of predicting thehydrophobicity of peptide sequences and secondary structureconformations such as alpha helices are well-known in the art, forinstance Pepfold and Pepwheel.

As used herein, hydrophobic amino acids are considered primarily toinclude amino acid residues, such as Ile (I), Leu (L), Val (V), Met (M),Phe (F), Tyr (Y), Ala (A), Trp (W). Polar uncharged amino acids areconsidered primarily to include amino acids such as Gln (Q), Asn (N),Thr (T), Ser (S), and Cys (C). Charged amino acids are consideredprimarily to include amino acids such as Asp (D), Glu (E), Arg (R), Lys(K), and His (H). Proline and glycine are considered neutral amino acidsand are not assigned to a specific group.

Proline tends to break or kink helices because it cannot donate an amidehydrogen bond (having no amide hydrogen), and because its side chaininterferes sterically. Its ring structure also restricts its backbonedihedral angle to the vicinity of −70°, which is less common inα-helices. One of skill understands that although proline may be presentat certain positions in the BMC-targeting signal polypeptides describedherein, the presence of more than three prolines within the sequencewould be expected to disrupt the helical structure. Accordingly, theBMC-targeting signal polypeptides of the invention preferably do notcomprise more than three prolines, more preferably do not comprise morethan two prolines within the alpha-helix forming sequence thereof.

Preferably, the BMC-targeting signal polypeptides are capable of formingcoiled coils in solution. Coiled coils are a well-known structuralconformation comprising two or more alpha helices coiled together in amanner akin to the strands of a rope. Preferably, the BMC-targetingsignal polypeptides are capable of forming coiled coil dimers insolution. The alpha helices within a coiled coil may be arranged in aparallel or anti-parallel conformation. Preferably, the coiled coil hasa left-handed conformation, although it may instead have a right-handedconformation. Method of determining the existence of coiled coilconformations are well-known in the art, for instance CCBuilder v1.0available at http://coiledcoils.chm.bris.ac.uk/app/cc_builder/.

The BMC-targeting polypeptide may be of any length. Preferably, theBMC-targeting signal polypeptide is 5 to 70 amino acids in length, morepreferably 10 to 22 amino acids in length, most preferably 14 to 20amino acids in length. Preferably, the BMC-targeting polypeptidecomprises an alpha-helical region that is at least 7 amino acids inlength.

As discussed above, naturally occurring BMC-targeting signalpolypeptides are found within proteins, typically enzymes, that localiseto BMCs. Typically, they are N-terminal sequences; they may however beC-terminal sequences, and in rare instances they are not N- orC-terminal sequences but rather are located within the interior of thenaturally occurring protein sequence.

Preferably, the BMC-targeting signal polypeptide of the presentinvention comprises the N-terminal 70, more preferably 60, morepreferably 50, 40, 30, 25, 20, 19, 18, 17, or 16 amino acids of anaturally occurring protein that comprises an N-terminal BMC-targetingsignal polypeptide. Alternatively preferably, the BMC-targeting signalpolypeptide of the present invention comprises the C-terminal 70, morepreferably 60, more preferably 50, 40, 30, 25, 20, 19, 18, 17, or 16amino acids of a naturally occurring protein that comprises a C-terminalBMC-targeting signal polypeptide.

Preferred naturally occurring proteins that include BMC-targeting signalpolypeptides are PduD and PduP preferably from Citrobacter freundii,Propionibacterium acnes, Fusobacterium ulcerans, Escherichia coli,Pectobacterium wasabiae, Listeria monocytogenes, Shewanella sp,Tolumonas aurensis, Yersinia frederiksenii, Klebsiella pneumoniae,Salmonella typhimurium, Salmonella enterica Paratyphi B str. andCitrobacter koseri, more preferably from Citrobacter freundii. In all ofthese proteins, the BMC-targeting signal polypeptide is an N-terminalsequence.

Preferably, the BMC-targeting signal polypeptide of the presentinvention comprises the N-terminal 70, more preferably 60, morepreferably 50, 40, 30, 25, 20, 19, 18, 17, or 16 amino acids of anaturally occurring PduP or PduD protein from an organism that naturallyexpresses Pdu BMCs, preferably from those microorganisms listed above,more preferably from Citrobacter freundii.

Preferably, the BMC-targeting signal polypeptide of the presentinvention comprises residues 1 to 16, more preferably residues 1 to 18of PduP from an organism that naturally expresses Pdu BMCs, preferablyfrom those microorganisms listed above, more preferably from Citrobacterfreundii, or residues 1 to 18 of PduD from an organism that naturallyexpresses Pdu BMCs, preferably from those microorganisms listed above,more preferably from Citrobacter freundii.

Preferably, the BMC-targeting signal polypeptide comprises the followingsequence:

X₁X₂X₃X₄X₅X₆X₇X₈X₉

wherein:

X₁, X₄, X₅, X₈, and X₉, are hydrophobic amino acids;

X₂, X₃ and X₆ are each independently polar or charged amino acids; and

X₇ is any amino acid. (SEQ ID NO: 76)

Preferably, X₁, X₄, X₅, X₈, and X₉ are each independently hydrophobicamino acids selected from the group consisting of I, L, V, M, F, Y, Aand W;

X₂, X₃ and X₆ are each independently polar or charged amino acidsselected from the group consisting of Q, N, T, S, C, D, E, R, K and H;and

X₇ is any amino acid. (SEQ ID NO: 77)

Preferably, X₁, X₄, X₅, X₈, and X₉ are each independently hydrophobicamino acids selected from the group consisting of I, L, V, M, and A;

X₂, X₃ and X₆ are each independently polar or charged amino acidsselected from the group consisting of Q, T, E, R, S, D and K; and

X₇ is any amino acid. (SEQ ID NO: 78)

Preferably, X₁ is a hydrophobic amino acid selected from the groupconsisting of I, L, V and A;

X₂ is a polar or charged amino acid selected from the group consistingof E, R and Q;

X₃ is a polar or charged amino acid selected from the group consistingof T, Q, E, S, D and K;

X₄ is a hydrophobic amino acid selected from the group consisting of I,L, V and M;

X₅ is a hydrophobic amino acid selected from the group consisting of I,L and V;

X₆ is a polar or charged amino acid selected from the group consistingof R, K, Q and E;

X₇ is any amino acid;

X₈ is a hydrophobic amino acid selected from the group consisting of I,L, V and A; and

X₉ is a hydrophobic amino acid selected from the group consisting of I,V, L and M (SEQ ID NO: 79).

Preferably, the BMC-targeting signal polypeptide comprises a sequenceselected from the group consisting of LEQIIRDVL (SEQ ID NO:1), LETLIRTIL(SEQ ID NO:2), LETLIRNIL (SEQ ID NO:3), LRQIIEDVL (SEQ ID NO:4),IEEIVRSVM (SEQ ID NO:5), IEQWKAVL (SEQ ID NO:6), VEKLVRQAI (SEQ IDNO:7), IQEIVRTLI (SEQ ID NO:8), VEEIVKRIM (SEQ ID NO:9), IESMVRDVL (SEQID NO:10), VQDIIKNW (SEQ ID NO:11), IRQWQEVL (SEQ ID NO:12), VRSWEEW(SEQ ID NO:13) and ARDLLKQIL (SEQ ID NO:14) or a variant thereof, morepreferably LEQIIRDVL (SEQ ID NO: 1) or a variant thereof, or LETLIRNIL(SEQ ID NO: 3) or LRQIIEDVL (SEQ ID NO: 4) or a variant thereof, mostpreferably LETLIRNIL (SEQ ID NO:3) or LRQIIEDVL (SEQ ID NO: 4) or avariant thereof.

Preferably, the BMC-targeting signal polypeptide comprises a sequenceselected from the group consisting of the sequences shown in the tablebelow:

SEQ ID NO: Sequence 15 (V/I)(V/Y)G(Q/K)(V/A/G/E)(Y/S/Q)(I/V/L/F)(N/Q/S/L)(K/Q/R)(M/L)(L/M/R)(V/L/C/Q) (T/S)(L/M)FP(H/D/E)(R/N/Q) 16(L/F)(S/P/A)(P/V)(E/Q)Q(A/S/Q/W)(Q/E/R) RIY(R/Q)G(S/N) 17M(D/N)(E/Q)(K/Q)(Q/E)(L/I)(K/R/E)(E/D)(I/M)(V/I)(R/E)(S/Q)(V/I)(L/M)A(E/Q/S) 18(A/K/S)(E/D)(A/E)L(I/V)(E/D/N)(L/E/S)(I/L)(V/I)(R/K/E/Q)(K/R)VL(E/A)(E/K)L 19MEI(N/D/T)E(K/E)(L/V)(L/V)(R/E)Q(I/V) (I/V)(E/K/A)(D/E)VL(K/S/R/A)(E/D)20 (M/I)(N/D)(T/E)(D/K)(A/L)(I/L)E(S/E)(M/I)V(R/K)(D/E/Q)VL(S,N)(M/L)(N/E/G)S 21M(N/D/E)(T/S/E)(S/L)E(L/V)E(T/Q/K/D)(L/I)(I/V)(R/K)(T/N/K)(I/V)(L/I)(S/L/R/N)E 22(A/P)(K/G)(S/Q)(S/D)(L/A)(T/N)E(E/Q)(D/Q)(I/V)Y(D/E)AVK(K/R)(V/I)(L/I)(E/G)(Q/E/S) (H/S)G(A/S)LD(P/V) 23MN(D/T)(I/T)(E/Q)(I/L)(A/E)(Q/N)(A/M)(V/I)(S/R/A)(T/K/N)IL(S/A/E/R)(D/K) (N/F/Y)(T/L/G)K 24LD(A/E)ES(A/V)(A/G)D(M/I)(T/A)E(M/Q)I (A/L)K(E/G)(L/M)(K/Q)(E/D)AG 25(D/P)(D/N)(A/E)(D/E/A)L(V/I)A(E/A/S)IT (K/R)(K/R/Q)V(M/L)(A/E)QL(G/K) 26VNEQ(L/M)VQDIV(Q/R/K)EVVA(K/R)MQI(S/T) 27DQE(A/Q)LV(K/Q)(A/L)IT(D/E)(Q/R/E)VMA (A/E)L(K/S)K 28MQ(I/A)(D/T)EE(L/A)IRSVV(A/Q)(Q/E)VL(A/S) (E/Q)(V/L)(G/N) 29(E/Q/D)(N/E/D)(V/I/L)(E/Q/A)(R/Q/D)(I/L/V)(I/L/V)(K/R/N)(E/Q/K)(V/I/L)(L/I/V)(E/Q/G) (Q/R/A)(L/M)(K/G/S) 30M(A/D)(K/I/N/L)(R/Y/)(E/N/S/L/F)(T/S)(P/N)(R/K)(V/L/F)(K/A)(E/V/M)(L/A)(A/T)(E/K) (R/N)(L/M) 31I(E/D/G)ALR(A/E/D)ELR(A/R)L(V/I)(V/A)EEL (A/R)(Q/E)L(I/N/G)(K/R)(R/Q)

More preferably, the BMC-targeting signal polypeptide comprises asequence selected from the group consisting of SEQ ID NO: 15, 17, 18,19, 20, 21, 22, 23, 26, 28 and 29.

More preferably, the BMC-targeting signal polypeptide comprises asequence selected from the group consisting of SEQ ID NO:19, SEQ IDNO:20 and SEQ ID NO:21. Still more preferably, the BMC-targeting signalpolypeptide comprises a sequence selected from SEQ ID NO: 19 and SEQ IDNO:21.

Preferably, the BMC-targeting signal polypeptide comprises a sequenceselected from the group consisting of the sequences shown in the tablebelow:

SEQ ID NO: Sequence 32 (V/I)(V/Y)G(Q/K)(V/A/G/E)(Y/S/Q)(I/V/L/F)(N/Q/S)(K/Q/R)(M/L)(L/M)(C/Q)(T/S)(L/M)FP (H/D/E)(R/N/Q) 17M(D/N)(E/Q)(K/Q)(Q/E)(L/I)(K/R/E)(E/D)(I/M)(V/I)(R/E)(S/Q)(V/I)(L/M)A(E/Q/S) 33(A/K/S)(E/D)(A/E)L(I/V)(E/D/N)(E/S)(I/L)(V/I)(R/K/E/Q)(K/R)VL(E/A)(E/K)L 34MEI(N/D/T)E(K/E)(L/V)(L/V)(R/E)Q(I/V)(I/V)(E/K)(D/E)VL(K/S/R/A)(E/D)(M/L) 20(M/I)(N/D)(T/E)(D/K)(A/L)(I/L)E(S/E)(M/I)V(R/K)(D/E/Q)VL(S,N)(M/L)(N/E/G)S 21M(N/D/E)(T/S/E)(S/L)E(L/V)E(T/Q/K/D)(L/I)(I/V)(R/K)(T/N/K)(I/V)(L/I)(S/L/R/N)E 22(A/P)(K/G)(S/Q)(S/D)(L/A)(T/N)E(E/Q)(D/Q)(I/V)Y(D/E)AVK(K/R)(V/I)(L/I)(E/G)(Q/E/S) (H/S)G(A/S)LD(P/V) 35MN(D/T)(I/T)(E/Q)(I/L)(E)(Q/N)(A/M)(V/I)(S/R)(T/K/N)IL(S/A/E/R)(D/K)(N/F/Y) (T/L/G)K 26VNEQ(L/M)VQDIV(Q/R/K)EVVA(K/R)MQI(S/T) 36MQ(I/A)(D/T)EE(L/A)IRSVVQ(Q/E)VL(A/S) (E/Q)(V/L)(G/N) 37(E/Q/D)(N/E/D)(V/I/L)(E/Q)(R/Q/D)(I/L/V)(I/L/V)(K/R/N)(E/Q/K)(V/I/L)(L/I/V)(E/Q/G) (Q/R/A)(L/M)(K/G/S)

More preferably, the BMC-targeting signal polypeptide comprises asequence selected from the group consisting of SEQ ID NO: 34, SEQ IDNO:20 and SEQ ID NO:21. Still more preferably, the BMC-targeting signalpolypeptide comprises a sequence selected from SEQ ID NO: 34 and SEQ IDNO:21.

Preferably, the BMC-targeting signal polypeptide comprises a sequenceselected from the group consisting of the sequences shown in the tablebelow, or a variant thereof:

SEQ ID NO: Sequence 38 VYGKEQFLRMRQSMFPDR 39 LAPEQQQRIYRGN 40MDQKQIEEIVRSVMAS 41 MNQQDIEQVVKAVLLKM 42 NTELVEEIVKRIMKQL 43MEINEKLLRQIIEDVLRDM 44 MEINEKLLRQIIEDVLRD 45 MEINEKLLRQIIEDVLSE 46MNTDAIESMVRDVLSRMNS 47 MNTSELETLIRTILSE 48 MNTSELETLIRNILSE 49MNTSELETLIRNILSEQL 50 AGTNYTEEQVFAAVKKVLNSSGSTDV 51 MVAKAIRDHAGTAQPSGNA52 IDIILAQQITVQIVKELKERG 53 DNADLVASITRKVMEQLG 54 VNEQLVQDIIKNVVASMQLT55 EPEDNEDVQAIVKAIMAKLNL 56 DTEMLVKMITEQVMAALKK 57 MQATEQAIRQVVQEVLAQLN58 EVEALVQRLTEEILRQLQ 59 IDETLVRSVVEEVVRAF 60 EDARDLLKQILQALS 61MDIREFSNKFVEATKNM 62 LDALRAELRALVVEELAQLIKR 63 MALREDRIAEIVERVLARL

Preferably, the BMC-targeting signal polypeptide comprises a sequenceselected from the group consisting of SEQ ID Nos: 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 54, 57, 59 and 60 or a variant thereof.

Preferably, the BMC-targeting signal polypeptide comprises a sequenceselected from the group consisting of SEQ ID NOs: 43, 44, 45, 47, 48 and49 or a variant thereof. More preferably, the BMC-targeting signalpolypeptide comprises a sequence selected from the group consisting of45, 48 or 49 or a variant thereof.

The microorganisms of the invention may comprise variants of any nucleicacid or polypeptide sequence disclosed herein, e.g. variants of thedisclosed BMC-targeting signal polypeptides. By “variant” is meant asequence with at least 75% identity (sequence identity) to the sequencedisclosed herein. e.g. to the sequence of a disclosed BMC-targetingsignal polypeptide. As used herein, variants retain the same function asthe nucleic acid or polypeptide of which they are a variant. VariantBMC-targeting signal polypeptides have the function of directing thepolypeptide to which they are attached to a BMC, if present.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. To determinethe percent sequence identity of two amino acid sequences, the sequencesare aligned for optimal comparison purposes (e.g., gaps can beintroduced in the sequence of one polypeptide for optimal alignment withthe other polypeptide). The amino acid residues at corresponding aminoacid positions are then compared. When a position in one sequence isoccupied by the same amino acid residue as the corresponding position inthe other sequence, then the molecules are identical at that position.“Identity” and “similarity” can be readily calculated by known methods,such as but not limited to Clustal and BLAST.

Preferably, a variant sequence has at least 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identity to the sequence to which it is compared, e.g. to theBMC-targeting signal polypeptides disclosed herein.

Preferably, a variant amino acid sequence as referred to hereincomprises no more than 6, more preferably no more than 5, morepreferably no more than 4, more preferably no more than 3, morepreferably no more than 2, most preferably no more than 1 mismatch(es)with the sequence to which it is compared, e.g. to a BMC-targetingsignal polypeptide disclosed herein. A mismatch is a non-identical aminoacid in the same position.

Preferably the variant sequences comprise only conservativesubstitutions as compared to the original amino acid sequence. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. In the context of the present invention, a conservative aminoacid substitution is the replacement of a hydrophobic amino acid withanother hydrophobic amino acid, the replacement of a polar amino acidwith another polar amino acid or the replacement of a charged amino acidwith another charged amino acid.

The microorganism of the present invention is essentially free of BMCs.As used herein, the term “essentially free of BMCs” means essentiallyfree of functional BMCs. A functional BMC is a BMC with the morphologyas seen in a microorganism that naturally expresses the BMC. Suchorganisms are well-known in the art and the morphology of naturallyoccurring functional BMCs is well-characterised.

BMCs are functional when they have an intact capsule, i.e. a closedshell. Such a morphology is termed a closed capsule morphology herein.Disruptions in BMC formation can lead to improperly formed BMCs that arenot fully closed. Functional and non-functional BMCs, i.e. those withand without the correct closed capsule morphology can be readilyidentified by a number of techniques known in the art, e.g. bytransmission electron microscopy. Preferably, the microorganism of theinvention is essentially free of closed BMCs. Preferably themicroorganism of the invention is essentially free of functional andimproperly formed BMCs, i.e. those with and without a closed capsulemorphology, i.e. closed and un-closed shells.

Preferably, the microorganism is essentially free of one or more,preferably all of Pdu, Eut and carboxysome BMCs. As mentioned above,preferably, the microorganism of the invention does not naturallyexpress, i.e. comprise, BMCs. Preferably, the microorganism of theinvention does not naturally express any BMCs.

Preferably, the microorganism is of a species or strain that does notnaturally express BMCs. Alternatively viewed, the microorganism of theinvention is preferably of a species or strain that lacks the genesnecessary for the formation of BMCs. Such microorganisms are entirelyfree of BMCs, i.e. they lack BMCs. In the case of microorganism speciesor strains that do not produce BMCs naturally, the microorganisms of thepresent invention of the same species or strains are entirely free ofsaid BMCs, and this is achieved simply by not genetically engineeringthe microorganisms to provide BMC production capability. Clearly,microorganisms that are not bacteria lack any BMCs. Furthermore, thevast majority of sequenced bacterial species lack BMCs.

In an alternative preferred embodiment, the microorganism of theinvention is of a species or strain that naturally expresses BMCs buthas been modified to reduce, i.e. it comprises modifications thatreduce, essentially all of the cell's ability to express BMCs. In otherwords, the microorganism of the invention is preferably of a species orstrain that naturally expresses BMCs but has been modified to reduceessentially all of the ability to express BMCs. Thus, in suchmicroorganisms, the native ability of the cell to express (i.e. produce)BMCs is interrupted, inhibited, or deleted, i.e. such that it expressesno more than de minimis level of BMCs.

Alternatively viewed, preferably the microorganism of the invention isof a species or strain that naturally comprises BMCs but has beengenetically modified to inhibit essentially all BMC formation. In otherwords, the microorganism is of a species or strain that possesses thegenes necessary for the formation of BMCs but has been modified suchthat essentially all BMC formation is inhibited, interrupted or deleted,i.e. such that it produces no more than de minimis level of BMCs.

In the embodiments in which the microorganism of the invention is of astrain that naturally comprises the genes necessary for the expressionof BMCs, the microorganism of the invention preferably comprises a lossof function mutation in one or more of said genes. Preferably, themicroorganism comprises loss of function mutations in at least one geneencoding a protein comprising a BMC domain and at least one geneencoding a protein comprising a bacterial microcompartment vertexdomain. Preferably, the microorganism comprises a loss of functionmutation in the regulatory region of the BMC operon(s) that are presentin the naturally occurring microorganism strain, preferably in theoperon's promoter.

In the embodiments in which the microorganism of the invention is of astrain that naturally comprises the genes necessary for the expressionof BMCs, the microorganism of the invention preferably comprises adisruption in one or more of said genes. Preferably, the microorganismcomprises a disruption in at least one gene encoding a proteincomprising a BMC domain and at least one gene encoding a proteincomprising a bacterial microcompartment vertex domain. Preferably, themicroorganism comprises a disruption in the regulatory region of the BMCoperon(s) that are present in the naturally occurring microorganismstrain, preferably in the operon's promoter.

As mentioned above, a loss of function mutation is a disruption of agene. A loss of function mutation, i.e. disruption, may comprisecomplete or partial inactivation of the gene, for instance by missensemutations, nonsense mutations, insertions, deletions, duplications,frameshift mutations, repeat expansions and any combination thereof.

Thorough analyses of bacterial genomes have been performed to identifyspecies that comprise BMC genes (US 2012/0210459, Axen et al., (2014)PLOS Computational Biology 10(10):e1003898 and Jorda J, et al., (2013)Protein Science: A Publication of the Protein Society. 22(2):179-195).It was determined that 23 different types of BMCs were encoded in 30distinct locus (sub)types found in 23 bacterial phyla. Dataset S1 ofAxen et al. comprehensively lists sequenced bacterial species thatnaturally comprise BMCs.

By “essentially free of BMCs” is meant that the microorganism is inessence free of BMCs but it does not mean that there is a strictrequirement for the microorganism to lack BMCs entirely. There ispotential for a de minimis level of BMC production even after steps havebeen taken to inhibit the production of BMCs. This is becausemicroorganisms are biological systems that cannot be as preciselycontrolled as, for instance, mechanical or chemical systems. Forinstance, random genetic mutation could, in rare instances, lead to theexpression of BMC capsule proteins. While a detailed inspection mayreveal that some BMCs are present, they are present in such smallquantities that for the purposes intended they can be considered absent.The microorganism of the invention is essentially free of BMCs if it hasbeen genetically modified to inhibit essentially all BMC formation.Preferably, the microorganism is free, i.e. entirely free of BMCs.

In any case, the level of BMC production in the microorganisms of thepresent invention is essentially zero. In embodiments in which themicroorganism is of a strain that naturally produces BMCs, the level ofBMC production in the microorganism of the present invention ispreferably at most 10%, more preferably at most 5%, still morepreferably at most 1% of the level of BMC production in the same strainthat has not been modified according to the invention. The person ofordinary skill in the art will readily be able to determine the extentof BMC production in a cell or population of cells, for instance bytransmission electron microscopy of sectioned cells, and quantifyingrelative protein levels by SDS gel electrophoresis.

Genetically engineering a microorganism to be essentially free of one ormore types of BMC would be within the competencies of one of ordinaryskill in the art and any suitable approach may be used. The genesrequired for BMC formation are well characterised, for instance in US2012/0210459, Chowdhury et al., (2014) Microbiol. Mol. Biol. Rev. 78(3):438, and Axen et al., (2014) PLOS Computational Biology 10(10):e1003898.

Preferably, one or more of the proteins required for BMC formation aredown-regulated in the microorganism of the present invention. Bydown-regulated is meant that the level of expression of said protein isin the microorganism of the invention is at most 10%, preferably at most5%, more preferably at most 1% of the level of expression of saidprotein in a microorganism of the same strain that has not been modifiedaccording to the invention. As used herein, down-regulation of a proteinis equivalent to down-regulation of a gene encoding that protein, andvice versa. Most preferably, the microorganism of the invention lacksthe one or more proteins required for BMC formation, i.e. themicroorganism is one in which the protein or the gene encoding saidprotein has been eliminated. Most preferably, the microorganism of theinvention lacks the one or more genes required for BMC formation, i.e.the microorganism is one in which the one or more genes required for BMCformation have been eliminated.

Genetic engineering techniques for down-regulating and eliminating theexpression of a gene/protein of interest are well-known in the art andany such technique may be used in the context of the present invention.Preferably, the microorganism of the invention comprises a deletion,interruption or deleterious mutation in one or more of the genesencoding a protein required for BMC formation that results in thereduction or elimination of expression of said protein. Alternatively,the relevant gene may be silenced using a short DNA or RNAoligonucleotide that has a sequence complementary to either gene or anmRNA transcript, e.g. antisense oligonucleotides.

The down-regulated protein required for formation of functional BMCs(i.e. BMCs having an intact, closed capsule) is preferably either:

i) a protein comprising a BMC-domain. A BMC domain is a domain common tothe majority of known BMC shell proteins. The BMC domains are typicallyflat hexamers that tile edge to edge to form extended protein sheets; orii) a pentameric bacterial microcompartment vertex (BMV) protein. Theseproteins are non-BMC-domain shell proteins that form the vertices of theBMC capsule.

Both BMC-domain containing proteins and BMV proteins are required forthe formation of intact, closed BMCs. Preferably, the microorganism ofthe present invention is essentially free of closed BMCs.

Preferably, the expression of one or more proteins comprising a BMCdomain is down-regulated in the microorganism of the present invention.Preferably the expression of one or more BMV proteins is down-regulatedin the microorganism of the present invention. Preferably, theexpression of at least one protein comprising a BMC domain and at leastone protein comprising a BMV domain is down-regulated in themicroorganism of the invention.

As mentioned above, a number of types of BMCs are known in the art,including Pdu BMCs, Eut BMCs, and carboxysomes. Throughout, “Pdu” standsfor “propanediol utilization” and Eut” stands for “ethanolamineutilization”. In nature, BMC proteins are typically expressed from thesame operon as the proteins, e.g. enzymes, that comprise BMC-targetingpolypeptides and that localise within the BMCs. For example, in the Pduoperon, the Pdu BMC proteins are expressed from the same operon as thePdu enzymes. Some naturally occurring microorganisms express more thanone type of BMC. For instance, Salmonella possesses the genes necessaryfor expression of both Pdu and Eut BMCs.

In bacteria that naturally express Pdu BMCs, it is known that theformation of functional BMCs with closed capsules requires at least theexpression of the BMC shell proteins PduA, B, B′, J, K, M, N, T and U.Preferably, the expression of PduA, B, B′, J, K, M, N, T or U or anycombination thereof is down-regulated in the microorganisms of thepresent invention. Preferably, the expression of PduN is down-regulated.PduN is known to be the BMV protein of the closed Pdu BMC and it hasbeen shown previously that PduN deletion mutants form grossly abnormal,non-functional BMCs. Preferably the expression of PduB and PduB′ aredown-regulated. PduBB′ deletion mutants have been shown previously to beunable to form BMCs. Preferably the expression of PduJ isdown-regulated. PduJ deletion mutants have been shown previously to beunable to form functional BMCs.

Preferably, the expression of PduN, B, B′, J, M or A or any combinationthereof is down-regulated. Preferably, the expression of PduN, B, B′, Jor A or any combination thereof is down-regulated, particularlypreferably when the microorganism is Salmonella. Preferably, theexpression of PduN and any one or more of Pdu B, B′, J, M and A isdown-regulated. Preferably, the expression of PduN, B, B′, J, M and A isdown-regulated. Preferably, the expression of PduA, B, B′, J, K, N, Tand U is down-regulated. Preferably, the expression of PduN, B, B′, Jand A is down-regulated, particularly preferably when the microorganismis Salmonella. Preferably, the expression of PduA, B, B′, J, K, M, N, Tand U is down-regulated.

Preferably, the expression of pduB, pduB′, pduJ, or pduN or anycombination thereof is down-regulated, most preferably the expression ofpduB, pduB′, pduJ, and pduN is down-regulated.

In bacteria that naturally express Eut BMCs, it is known that theformation of functional, closed BMCs requires at least the expression ofthe BMC shell proteins EutK, M, S, L and N. Preferably, the expressionof EutK, M, S, L or N or any combination thereof is down-regulated inthe microorganisms of the present invention. Preferably, the expressionof EutN is down-regulated. EutN is known to be the BMV protein of theclosed Eut BMC. Preferably, the expression of EutN and any one or moreof EutK, M, S and L is down-regulated. Preferably, the expression ofEutN, EutM, Eut S and EutL are down-regulated. Preferably, theexpression of EutK, M, S, L and N are down-regulated.

In bacteria that naturally express the alpha-carboxysome, it is knownthat the formation of functional, closed BMCs requires at least theexpression of the proteins CsoS1 A-D, CsoS2 and CsoS4. Preferably, theexpression of CsoS1 A-D, CsoS2 or CsoS4 is down-regulated. Preferably,the expression of CsoS1 A-D and CsoS2 is down-regulated. Preferably, theexpression of CsoS1 A-D and CsoS4 is down-regulated. Preferably, theexpression of CsoS2 and CsoS4 is down-regulated. Preferably, theexpression of CsoS1 A-D, CsoS2 and CsoS4 is down-regulated.

In bacteria that naturally express the beta-carboxysome, it is knownthat the formation of functional, closed BMCs requires at least theexpression of the proteins CcmK2, CcmO and CcmL. Preferably, theexpression of CcmK2, CcmO or CcmL is down-regulated. Preferably theexpression of CcmK2 and CcmO is down-regulated. Preferably theexpression of CcmK2 and CcmL is down-regulated. Preferably theexpression of CcmO and CcmL is down-regulated. Preferably the expressionof CcmK2, CcmO and CcmL is down-regulated. Preferably the expression ofCcmK1, 3 and 4 are also down-regulated.

The glycyl radical enzyme(GRM)-associated bacterial microcompartmentscan vary in the number and type of shell proteins in the operon. Theseshell proteins are homologues to the shell proteins of the other BMCsystems and belong to the same protein families. Like the Pdu BMC, theGRM BMC comprise s hexamers and pentamers, and the pentamers form thevertices of the BMC capsule. In bacteria that naturally express the GRMBMC, preferably the pentameric protein (Pfam03319) is down-regulated.

Preferably, if a microorganism of the invention is of a species thatnaturally expresses a particular BMCs type or types, then themicroorganism of the invention comprises a deletion or deleteriousmutation in a regulatory region of the operon for the production of saidBMC type or types, preferably in the promoter. Preferably, if amicroorganism of the invention is of a species that naturally expressesa particular BMCs type or types, then the microorganism of the inventioncomprises a deletion of the operon for the production of said BMC typeor types. In other words, if a microorganism of the invention is of aspecies that naturally expresses a particular BMCs type or types, thenthe microorganism of the invention is preferably a BMC null mutant. ABMC null mutant is a microorganism that has been modified such that itis devoid of any endogenous genes for the production of BMCs.

As mentioned above, in nature, microorganisms that naturally expressBMCs typically do so only under certain conditions, namely in thepresence of the substrate for the pathway that comprises steps catalysedby enzymes located within the BMCs. For instance, Pdu BMCs are onlyexpressed by microorganisms comprising the necessary genes when saidmicroorganisms are exposed to 1,2-propanediol. Similarly, Eut BMCs areonly expressed by microorganisms comprising the necessary genes whensaid microorganisms are exposed to ethanolamine.

Thus, in an alternative embodiment, the microorganism of the inventionis of a species or strain that naturally expresses BMCs, i.e. thatcomprises the genes necessary for the expression of BMCs, but whereinsaid microorganism is in an environment, e.g. a culture medium, thatdoes not permit expression of BMCs. In other words, preferably themicroorganism is present in a culture medium that lacks the molecule(s)that induce(s) the expression of BMCs in the microorganism. If themicroorganism naturally expresses Pdu BMCs, then said molecule is1,2-propanediol. If the microorganism expresses Eut BMCs, then saidmolecule is ethanolamine. The use of such limited culture media mayremove the need for genetic modification of a microorganism to ensurelack of BMC expression.

In another aspect, the present invention provides a method of producinga genetically modified microorganism as described herein, said methodcomprising transforming a microorganism with one or more heterologousnucleic acid molecules together encoding at least three differentproteins, each protein comprising an enzymatic domain and a bacterialmicrocompartment-targeting signal polypeptide, wherein said enzymaticdomains catalyse different substrate to product conversions in the samemetabolic pathway, and wherein said microorganism is essentially free ofBMCs

The features and embodiments described above in relation to thegenetically modified microorganisms of the invention apply mutatismutandis to the methods of producing a genetically modifiedmicroorganism disclosed herein. The microorganisms, one or moreheterologous nucleic acid molecules, at least three proteins, enzymaticdomains, BMC-targeting signal polypeptides, substrate to productconversions, products of interest, and BMCs are as defined above.

In said method, preferably the microorganism is transformed with one ormore plasmids, vectors or transformation cassettes comprising said oneor more nucleic acid molecules. Preferably, said vector is an expressionvector, preferably also comprising a strong heterologous promoteroperatively linked to said one or more nucleic acid molecules.

If the microorganism is of a species or strain that naturally expressesBMCs, then the method comprises the step of genetically modifying themicroorganism to inhibit essentially all BMC formation. Such steps areas described above. Alternatively, the method comprises culturing themicroorganism only in an environment, e.g. a culture medium, that doesnot permit expression of BMCs, i.e. that lacks the molecule(s) thatinduce(s) the expression of BMCs in the microorganism.

Alternatively viewed, the present invention provides a method ofproducing a genetically modified microorganism as described herein, saidmethod comprising over-expressing in a microorganism one or moreheterologous nucleic acid molecules together encoding at least threedifferent proteins, each protein comprising an enzymatic domain and abacterial microcompartment-targeting signal polypeptide, wherein saidenzymatic domains catalyse different substrate to product conversions inthe same metabolic pathway, and wherein said microorganism isessentially free of BMCs

As explained above, preferably said microorganism is naturally free ofBMCs, i.e. is of a strain that does not naturally express BMCs. In analternative preferred embodiment however, the microorganism of theinvention is of a strain that naturally expresses BMCs, therefore, theabove methods preferably comprise a step of modifying the microorganismor its environment to inhibit its ability to produce said BMCs, asdescribed above. Said inhibitions are preferably complete inhibitions.

Preferably, the step of modifying the microorganism to inhibit itsability to produce said BMCs comprises the step of down-regulating oneor more of the proteins required for BMC formation. Preferably, the stepof modifying the microorganism to inhibit its ability to produce saidBMCs comprises the step of eliminating one or more of the proteinsrequired for BMC formation. Preferably, said steps comprise deleting,interruption or deleteriously mutating one or more of the genes encodinga protein required for BMC formation that results in the reduction orelimination of expression of said protein. Alternatively, the methodcomprises silencing the one or more genes using a short DNA or RNAoligonucleotide that has a sequence complementary to either gene or anmRNA transcript, e.g. antisense oligonucleotides.

Preferably, if a microorganism of the invention is of a species thatnaturally expresses a particular BMCs type or types, then themicroorganism of the invention comprises a deletion or deleteriousmutation in a regulatory region of the operon for the production of saidBMC type or types, preferably in the promoter. Preferably, if amicroorganism of the invention is of a species that naturally expressesa particular BMCs type or types, then the microorganism of the inventioncomprises a deletion of the operon for the production of said BMC typeor types. In other words, if a microorganism of the invention is of aspecies that naturally expresses a particular BMCs type or types, thenthe microorganism of the invention is preferably a BMC null mutant. ABMC null mutant is a microorganism that has been modified such that itis devoid of any endogenous genes for the production of BMCs.

The discussion of preferred down-regulated proteins in the context ofthe microorganisms of the invention applies mutatis mutandis to themethods of producing the microorganisms of the invention.

In another aspect, the present invention provides a method of producinga product of interest, said method comprising growing a geneticallymodified microorganism described herein under conditions wherein theproduct is produced and optionally recovering the product. Said methodscomprise growing the genetically modified microorganism under conditionsin which said at least three different proteins are expressed, andpreferably wherein said proteins together form aggregates.

The methods of the present invention comprise growing, i.e. culturing, amicroorganism of the present invention under conditions that produce theproduct of interest. Any such conditions can be used and the person ofordinary skill in the art will readily be able to select and optimisethe conditions for their specific purposes. Typically, the microorganismwill be grown in a culture medium. If the microorganism of the inventionis a strain that naturally expresses BMCs, then preferably the methoddoes not comprise the step of applying to the culture medium any inducermolecule(s) that induce the expression of said BMCs in saidmicroorganism. Preferably, the method does not comprise the step ofapplying propanediol, ethanolamine, choline, fucose or rhamnose.

The methods of the present invention thus comprise fermentation.“Fermentation” as used herein is the bulk growth of microorganisms on agrowth medium, with the aim of producing a specific product by ametabolic process. Although fermentation is optionally a process thatconverts sugar to acids, gases or alcohols, it is not limited to thesesubstrates or products as used herein.

Typically, the microorganisms of the present invention are grown infermentation media for production of a product of interest. Defined orsynthetic growth media may also be used and the appropriate medium forgrowth of a particular microorganism will be known by one skilled in theart of microbiology or fermentation science.

Fermentation media for production of the products of interest arewell-known in the art and the skilled person will readily be able todetermine a suitable fermentation media for their specific purpose. Anappropriate, or effective, fermentation medium refers to any medium inwhich a genetically modified microorganism of the present invention,when cultured, is capable of producing the product of interest. Such amedium is typically an aqueous medium comprising assimilable carbon,nitrogen and phosphate sources. Such a medium can also includeappropriate salts, minerals, metals and other nutrients.

Carbon sources are well-known in the art and the skilled person willreadily be able to determine the appropriate carbon source for themicroorganism species being used and the product of interest. It iscontemplated that the source of carbon utilized can encompass a widevariety of carbon containing substrates and will only be limited by thechoice of organism. Sources of assimilable carbon which can be used in asuitable fermentation medium include, but are not limited to, sugars andtheir polymers, including, dextrin, sucrose, maltose, lactose, glucose,fructose, mannose, sorbose, arabinose and xylose; fatty acids; organicacids such as acetate; primary alcohols such as ethanol and n-propanol;and polyalcohols such as glycerol. Preferred carbon sources includemonosaccharides, disaccharides, and trisaccharides. The most preferredcarbon source is glucose or glycerol. The concentration of a carbonsource in the fermentation medium should promote cell growth, but not beso high as to repress growth of the microorganism used.

Sources of assimilable nitrogen which can be used in a suitablefermentation medium include, but are not limited to, simple nitrogensources, organic nitrogen sources and complex nitrogen sources. Suchnitrogen sources include anhydrous ammonia, ammonium salts andsubstances of animal, vegetable and/or microbial origin. Suitablenitrogen sources include, but are not limited to, protein hydrolysates,microbial biomass hydrolysates, peptone, yeast extract, ammoniumsulfate, urea, and amino acids.

The fermentation medium can contain other compounds such as inorganicsalts, vitamins, trace metals or growth promoters. Such other compoundscan also be present in carbon, nitrogen or mineral sources in theeffective medium or can be added specifically to the medium. Thefermentation medium can also contain a suitable phosphate source. Suchphosphate sources include both inorganic and organic phosphate sources.Preferred phosphate sources include, but are not limited to, phosphatesalts such as mono or dibasic sodium and potassium phosphates, ammoniumphosphate and mixtures thereof. A suitable fermentation medium can alsoinclude a source of magnesium, preferably in the form of aphysiologically acceptable salt, such as magnesium sulfate heptahydrate,although other magnesium sources in concentrations which contributesimilar amounts of magnesium can be used.

The fermentation medium can also include a biologically acceptablechelating agent, such as the dihydrate of trisodium citrate. Thefermentation medium can also include a biologically acceptable calciumsource, including, but not limited to, calcium chloride. Thefermentation medium can also include sodium chloride. Preferably, theculture medium lacks the molecule(s) that induce(s) the expression ofBMCs in the microorganism. Alternatively viewed, the present inventionprovides a culture medium comprising a microorganism of the invention,wherein said culture medium lacks the molecule(s) that induce(s) theexpression of BMCs in the microorganism. Such molecules are describedabove. Preferably, the culture media lacks one or more, preferably allof propanediol, ethanolamine, choline, fucose and rhamnose.

The microorganisms of the invention described herein can be culturedusing standard laboratory or industrial techniques known in the art. Thegrowth of the microorganisms described herein can be measured by methodsknown in the art, for instance by measuring the optical density (OD) ofcell cultures over time.

The temperature of the fermentation medium can be any temperaturesuitable for growth and production of the product of interest. Forexample, prior to inoculation of the fermentation medium with aninoculum, the fermentation medium can be brought to and maintained at atemperature in the range of from about 20° C. to about 45° C.,preferably to a temperature in the range of from about 25° C. to about40° C.

The pH of the fermentation medium can be controlled by the addition ofacid or base to the fermentation medium. In such cases when ammonia isused to control pH, it also conveniently serves as a nitrogen source inthe fermentation medium. Preferably, the pH is maintained from about 3.0to about 9.0, more preferably from about 4 to about 8.0, still morepreferably from about 6.5 to about 7.5, most preferably about 7.4.

Fermentations can be performed under aerobic or anaerobic conditions.The fermentation medium can also be maintained to have a dissolvedoxygen content during the course of fermentation to maintain cell growthand to maintain cell metabolism for production of the product ofinterest. The oxygen concentration of the fermentation medium can bemonitored using known methods, such as through the use of an oxygenelectrode. Oxygen can be added to the fermentation medium using methodsknown in the art, for example through agitation and aeration of themedium by stirring or shaking. Preferably, the oxygen concentration inthe fermentation medium is in the range of from about 20% to about 100%of the saturation value of oxygen in the medium based upon thesolubility of oxygen in the fermentation medium at atmospheric pressureand at a temperature in the range of from about 20° C. to about 40° C.Periodic drops in the oxygen concentration below this range may occurduring fermentation, however, without adversely affecting thefermentation.

Although aeration of the medium has been described herein in relation tothe use of air, other sources of oxygen can be used. Particularly usefulis the use of an aerating gas which contains a volume fraction of oxygengreater than the volume fraction of oxygen in ambient air. In addition,such aerating gases can include other gases which do not negativelyaffect the fermentation.

Although the carbon source concentration can be maintained withindesired levels by addition of, for example, a substantially pure glucosesolution, it is acceptable, and may be preferred, to maintain the carbonsource concentration of the fermentation medium by addition of aliquotsof the original fermentation medium. The use of aliquots of the originalfermentation medium may be desirable because the concentrations of othernutrients in the medium (e.g. the nitrogen and phosphate sources) can bemaintained simultaneously.

The amount of product in the fermentation medium can be determined usinga number of methods known in the art, for example, high performanceliquid chromatography (HPLC) or gas chromatography (GC).

A batch method of fermentation can be used with the microorganismsdescribed herein. A classical batch fermentation is a closed systemwhere the composition of the medium is set at the beginning of thefermentation and not subject to artificial alterations during thefermentation. Thus, at the beginning of the fermentation the medium isinoculated with the desired organism or organisms, and fermentation ispermitted to occur without adding anything to the system. Typically,however, a “batch” fermentation is batch with respect to the addition ofcarbon source and attempts are often made at controlling factors such aspH and oxygen concentration. In batch systems the metabolite and biomasscompositions of the system change constantly up to the time thefermentation is stopped. Within batch cultures cells progress through astatic lag phase to a high growth log phase and finally to a stationaryphase where growth rate is diminished or halted. If untreated, cells inthe stationary phase will eventually die. Cells in log phase generallyare responsible for the bulk of production of end product orintermediate.

A Fed Batch system can also be used with the microorganisms describedherein. A Fed Batch system is similar to a typical batch system with theexception that the carbon source substrate is added in increments as thefermentation progresses. Fed Batch systems are useful when cataboliterepression (e.g. glucose repression) is apt to inhibit the metabolism ofthe cells and where it is desirable to have limited amounts of substratein the media. Measurement of the actual substrate concentration in FedBatch systems is difficult and is therefore estimated on the basis ofthe changes of measurable factors such as pH, dissolved oxygen and thepartial pressure of waste gases such as CO₂. Batch and Fed Batchfermentations are common and well known in the art.

Although a batch mode can be performed, it is also contemplated thatcontinuous fermentation methods could also be performed with themicroorganisms described herein. Continuous fermentation is an opensystem where a defined fermentation medium is added continuously to abioreactor and an equal amount of conditioned media is removedsimultaneously for processing. Continuous fermentation generallymaintains the cultures at a constant high density where cells areprimarily in log phase growth.

Continuous fermentation allows for the modulation of one factor or anynumber of factors that affect cell growth or end product concentration.For example, one method will maintain a limiting nutrient such as thecarbon source or nitrogen level at a fixed rate and allow all otherparameters to vary. In other systems a number of factors affectinggrowth can be altered continuously while the cell concentration,measured by media turbidity, is kept constant. Continuous systems striveto maintain steady state growth conditions and thus the cell loss due tothe medium being drawn off must be balanced against the cell growth ratein the fermentation. Methods of modulating nutrients and growth factorsfor continuous fermentation processes as well as techniques formaximizing the rate of product formation are well known in the art ofindustrial microbiology.

It is contemplated that the present invention can be practiced usingeither batch, fed batch or continuous processes and that any known modeof fermentation would be suitable. Additionally, it is contemplated thatcells can be immobilized on a substrate as whole cell catalysts andsubjected to fermentation conditions for production.

Products can be isolated from the fermentation medium by methods knownto one skilled in the art. For instance, solids may be removed from thefermentation medium by centrifugation, filtration, decantation, or thelike. Products of interest in solution may be isolated from thefermentation medium using methods such as distillation, azeotropicdistillation, liquid-liquid extraction, adsorption, gas stripping,membrane evaporation, pervaporation or vacuum flash fermentation.

In another aspect, the present invention comprises a cell free system,said system comprising aggregates comprising at least three differentproteins, each protein comprising an enzymatic domain and a bacterialmicrocompartment-targeting signal polypeptide, wherein said enzymaticdomains each catalyse a different substrate to product conversion in thesame metabolic pathway, and wherein said system does not comprisebacterial microcompartments.

The system is suitable for the production of a product of interest,wherein said enzymatic domains each catalyse a different substrate toproduct conversion in the same metabolic pathway for the production ofsaid product of interest.

The features and embodiments described above in relation to thegenetically modified microorganisms of the invention apply mutatismutandis to the cell free systems disclosed herein. The microorganisms,one or more heterologous nucleic acid molecules, at least threeproteins, enzymatic domains, BMC-targeting signal polypeptides,substrate to product conversions, products of interest, and BMCs are asdefined above.

The conditions of the cell free system preferably mimic in vivoconditions. It would be within the competencies of one of ordinary skillin the art to modify the conditions in the cell free system to suittheir particular purposes and the particular proteins employed.Preferably the pH of the system is one at which the enzymatic domainscan function and the proteins remain aggregated. Preferably, the systemcomprises a buffer. Preferably, the pH of the system is between 5 and 9.Suitable buffers are well-known in the art. For example, a potassiumphosphate buffer (pH 8) may be used, for instance at a concentration ofabout 100 mM.

Depending on the proteins, particularly the enzymatic domains present inthe system, the system may or may not comprise a salt solution.Preferably, the cell free system comprises said aggregates and anaqueous solution, preferably a salt solution, for instance comprisingNaCl and/or MgCl₂. Optionally, the NaCl is present at a concentration ofabout 100 mM. Optionally the MgCl₂ is present at a concentration ofabout 2.5 mM. If present, the solution is preferably an aqueoussolution.

Preferably, the system comprises the co-factors necessary for thecatalytic activity of the enzymatic domains of the at least threeproteins in the system, for instance NADH, for instance at aconcentration of about 0.1 mM. The skilled person will be aware of theappropriate cofactors for any particular enzymatic domain. Preferably,the system comprises ATP, for instance at a concentration of about 1 mM.

Preferably, the system comprises potassium phosphate buffer (pH 8),NaCl, NADH, MgCl2, McCl2 and ATP. Preferably, the system comprises about100 mM potassium phosphate buffer (pH 8), about 100 mM NaCl, about 0.1mM NADH, about 2.5 mM MgCl2, about 0.1 mM McCl2 and about 1 mM ATP.

Preferably, the system comprises cell lysate obtained from a populationof microorganisms of the present invention. Preferably, the cell freesystem is prepared by culturing the microorganism of the invention,suspending the cultured cells in an aqueous solution and lysing them toresult in a cell lysate. The cell lysate will comprise the desiredprotein aggregates since the aggregates will have formed in themicroorganism during the culturing step.

Optionally, one or more purification steps can be performed to removeunwanted cellular fractions and/or to selectively isolate theaggregates. For instance, exclusion fractionation/chromatography may beused and the aggregates isolated by centrifugation. Various methods ofcellular fractionation are well known in the art, as are methods for theisolation of proteins of interest including those in aggregated form.Suitable methods are disclosed, for instance, in Principles andTechniques of Practical Biochemistry (Wilson, K. & Walker, J) CambridgeUniversity Press 5^(th) Ed. Purification methods are disclosed, forinstance, in Rodriguez-Carmona et al. (2010) Microbial Cell Factories9:71.

It will be within the competencies of one of ordinary skill in the artto adjust the composition and conditions of the cell free system forhis/her intended purpose and depending on the nature of the at leastthree proteins therein. For instance, the skilled person will be awareof how the pH of a cell free system can be adjusted to ensure that theaggregated proteins remain aggregated and the enzymatic domains remainfunctional. Similar considerations will be given to the temperature andsalt concentration of the system.

The enzymatic domains present within the aggregated proteins in the cellfree system each catalyse a different substrate to product conversion inthe same metabolic pathway, i.e. in a pathway for the production of aproduct of interest. Preferably, the cell free system thereforecomprises a suitable level of substrate(s) for the production of theproduct of interest.

In another aspect, the present invention provides a method of producinga product of interest, said method comprising:

i) providing a cell free system comprising aggregates comprising atleast three different proteins, each protein comprising an enzymaticdomain and a bacterial microcompartment-targeting signal polypeptide,wherein said enzymatic domains each catalyse a different substrate toproduct conversion in the same metabolic pathway for the production ofthe product of interest, and wherein said system does not comprisebacterial microcompartments;ii) applying to said system the substrate of the first substrate toproduct conversion in the metabolic pathway that is catalysed by one ofsaid enzymatic domains; andiii) optionally recovering the product of interest.

The present invention provides a method of producing a product ofinterest, said method comprising:

i) growing a genetically modified microorganism described herein underconditions wherein said at least three proteins are expressed,preferably over-expressed, and wherein said proteins together formaggregates,ii) obtaining said aggregates from said microorganisms, andiii) using said aggregates in a cell free system for the production ofsaid product of interest, under conditions wherein the product isproduced;and optionally recovering the product.

The cell free system is as described herein. Preferably, the step ofobtaining said aggregates comprises lysing the microorganisms andobtaining the cell lysate, as described above.

As used herein, the singular forms “a,” “an” and “the” include pluralreferences unless the content clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a mixture of two or more cells.

The invention will now be further described in the followingnon-limiting Examples and the Figures in which:

FIG. 1 illustrates a pathway for the synthesis of 1,2-propanediol fromglycerol. Glycerol dehydrogenase and dihydroxyacetone kinase catalysethe conversion of glycerol to dihydroxyacetone phosphate, via theintermedia dihydroxyacetone. Methylglyoxal synthase catalyses theconversion of dihydroxyacetone phosphate to methylglyoxal. Glyceroldehydrogenase and 1,2-propanediol oxidoreductase catalyse the conversionof methylglyoxal to 1,2-propanediol, via the intermediate lactaldehyde.

FIG. 2 shows the specific activity of the enzymes involved in themicrobial synthesis of 1,2-propanediol: (a) glycerol dehydrogenase (b)dihydroxyacetone kinase (c) methylglyoxal synthase (d) 1,2-propanedioloxidoreductase, when untagged (i.e. not linked to a BMC-targeting signalsequence), when tagged with the BMC-targeting signal sequence D18 andwhen tagged with the BMC-targeting signal sequence P18.

FIG. 3 provides a statistical analysis showing the percentage of cellsthat contan inclusion bodies when said cells express either untaggedGldA, DhaK, MgsA or FucO, or P18- or D18-tagged versions of saidproteins.

FIG. 4 shows the results of a protease protection assay of GFP fused toa C-terminal proteolysis tag (SsrA) and an N-terminal tag being either aBMC targeting signal peptide (P18 or D18) or a non-targeting His-tag, inthe presence and absence of BMCs (AU). E. coli competent cells weretransformed with plasmids encoding the protein fusions with and withoutshell proteins and the resulting strains were cultured for 24 hours,samples were taken and run on a 15% denaturing polyacrylamide gel. Gelswere subsequently submitted to Western blotting using an anti-GFPprimary antibody. Total lysates were analysed by SDS-PAGE andsubsequently western blotted with an anti-GFP primary antibody. Celldensities were normalised to an OD₆₀₀=2.5 for loading of samples.

FIG. 5 shows in vivo 1,2-propanediol production. The graph shows the1,2-propanediol content (normalised to OD₆₀₀=1) over 96 h in the growthmedium of strains that lack shell proteins and 1,2-propanediol producingenzymes (●), Shell proteins only (◯), un-tagged 1,2-propanediolproducing enzymes (▾), 1,2-propanediol producing enzymes tagged withtargeting sequences (Δ), un-tagged 1,2-propanediol producing enzymes andshell proteins (▪), 1,2-propanediol producing enzymes tagged withtargeting sequences and shell proteins (□). Data points represent anaverage of three independent experiments; standard deviations arerepresented by error bars.

FIG. 6 shows in vivo 1,2-propanediol production. The graph shows the1,2-propanediol content (not normalised to OD₆₀₀=1) over 96 h in thegrowth medium in of strains that lack shell proteins and 1,2-propanediolproducing enzymes (●), Shell proteins only (◯), un-tagged1,2-propanediol producing enzymes (▾), 1,2-propanediol producing enzymestagged with targeting sequences (Δ), un-tagged 1,2-propanediol producingenzymes and shell proteins (▪), 1,2-propanediol producing enzymes taggedwith targeting sequences and shell proteins (□). Data points representan average of three independent experiments; standard deviations arerepresented by error bars.

FIG. 7 shows thin sections of E. coli strains labelled with an anti-hisantibody and then with a secondary antibody conjugated to 10 nm goldparticles viewed under TEM (a) Wild type (b) Shell proteins only (c)1,2-propanediol producing enzymes tagged with targeting sequences (d)1,2-propanediol producing enzymes tagged with targeting sequences andshell proteins (e) un-tagged 1,2-propanediol producing enzymes (f)un-tagged 1,2-propanediol producing enzymes and shell proteins.

FIG. 8 shows TEM analysis of strains expressing (A) His-tagged GldA (B)P18-tagged GldA (C) His-tagged DhaK (D) P18-tagged DhaK (E) His-taggedMgsA (F) D18-tagged MgsA (G) His-tagged FucO (H) D18-tagged FucO. Scalebar shows 0.2 μM.

EXAMPLES

In this study we are creating fusion proteins between Pdu targetingpeptides and the four 1,2-propanediol producing enzymes to target theenzymes to recombinant Pdu microcompartment shells. We explore how thetargeting peptides affect the activity of the different enzymes andtheir properties, particularly solubility. Strains are engineered forthe targeting of all enzymes to microcompartments and compared for their1,2-propanediol production to strains containing the native enzymes andalso to a strain containing enzymes with targeting peptides but no shellproteins. The protein solubility of these strains is investigated by TEManalysis and protein aggregation is found to play an unexpected butimportant role in the efficiency of our pathway. Finally, we propose analternative pathway engineering approach alongside compartmentalisationin protein shells.

Materials and Methods

Strains

The strains used in this study are shown in Table A below:

TABLE A Strains used in this study Strain Genotype Source BL21*(DE3) F-ompT hsdSB (rB- mB-) gal dcm (DE3) Novagen BL21*(DE3) F- ompT hsdSB(rB-mB-) gal dcm (DE3) Novagen pLysS pLysS (CamR)

The BL21*(DE3) strain comprises genes encoding Eut BMCs. To ensure thatEut BMCs were not produced by the microorganisms during this study,ethanolamine was not included in any fermentation media. The absence ofBMCs was confirmed by TEM.

Plasmid Construction

Plasmids were constructed to include each of the genes of interest withan N-terminal tag comprising a BMC-targeting signal polypeptide (“P18”or “D18”) and/or a hexa-histidine tag.

The genes of interest were the four enzymes of the metabolic pathway forthe production of 1,2-propanediol from glycerol, as outlined in FIG. 1,i.e. glycerol dehydrogenase (GldA), dihydroxyacetone kinase (DhaK),methylglyoxal synthase (MgsA) and 1,2-propanediol oxidoreductase (FucO).

The BMC-targeting signal polypeptide-containing tags used in the studywere as follows:

“D18”: (SEQ ID NO: 64) MEINEKLLRQIIEDVLSEPMGSSHHHHHHSSGLVPRGSH

(N-terminal 18 amino acids of PduD from Citrobacter freundii (theBMC-targeting signal polypeptide) followed by flexible linker PMGSS,6-his linker, flexible linker SSGL, thrombin cleavage site LVPRGS andamino acid linker H)

“P18”: (SEQ ID NO: 65) MNTSELETLIRNILSEQLAMGSSHHHHHHSSGLVPRGSH

(N-terminal 18 amino acids of PduP from Citrobacter freundii (theBMC-targeting signal polypeptide) followed by flexible linker AMGSS,6-his linker, flexible linker SSGL, thrombin cleavage site LVPRGS andamino acid linker H)

All primers used in this study are listed in Table B below.

TABLE B Oligonucleotide primers used in this study, restrictionsites are underlined Name Sequence 5′-3′ GldA_NdeI_FWCATCATATGGACCGCATTATTC AATCACC (SEQ ID NO: 66) GldA_SpeI_RVCATACTAGTTTATTCCCACTCT TGCAGG (SEQ ID NO: 67) dhaK_NdeI_FWCGCCATATGTCTCAATTCTTTT TTAACCAACGCACC (SEQ ID NO: 68) dhaK_SpeI_RVCATACTAGTTTAGCCCAGCTCA CTCTCCGC (SEQ ID NO: 69) mgsA_NdeI_FWCATCATATGGAACTGACGACTC GCACTTTACC (SEQ ID NO: 70) mgsA_SpeI_RVCATACTAGTTTACTTCAGACGG TCCGCGAG (SEQ ID NO: 71) fucO_NdeI_FWCCGCATATGGCTAACAGAATGA TTCTG (SEQ ID NO: 72) fucO_SpeI_RVCCTACTAGTTTACCAGGCGGTA TGG (SEQ ID NO: 73) GFP_NdeI_FWGTACATATGAGCAAAGGAGAAG AACTTTTC (SEQ ID NO: 74) GFP-SsrA_SpeI_RVGACACTAGTTTAAGCTGCTAAA GCGTAGTTTTCGTCGTTTGCTG CTTTGTACAGCTCATCCATGCC(SEQ ID NO: 75)

All genes were amplified with flanking Ndel and Spel restriction sitesand each was ligated into pET14b, pET14b-D18 and pET14b-P18 vectorsusing Ndel and Spel restriction sites.

Plasmids pML-1 to pML-6 as outlined in Table C, were constructed by a‘Link and Lock’ approach utilizing the compatible sticky ends formed bydigestion with Xbal and Spel (McGoldrick et al., (2005) J Biol Chem14:1086-1094).

TABLE C Plasmids used in this study Plasmid name Genotype DescriptionSource pET14b pET14b Overexpression vector Novagen containing N-terminalpolyhistidine-tag pET14b-D18 pET14b-D18 Overexpression vector This studycontaining an N-terminal D18 targeting tag and an N-terminalpolyhistidine-tag pET14b-P18 pET14b-P18 Overexpression vector This studycontaining an N-terminal P18 targeting tag and an N-terminalpolyhistidine-tag pLysS PlysS Overexpression vector NovagenpLysS-PduABB′JKNU pLysS-PduABB′JKNU Construct for expression Parsons etal., of empty Pdu BMC 2010 pET14b-gldA pET14b-gldA PCR product of gldAThis study ligated into Ndel/Spel sites of pET14b pET14b-dhaKpET14b-dhaK PCR product of dhaK This study ligated into Ndel/Spel sitesof pET14b pET14b-mgsA pET14b-mgsA PCR product of mgsA This study ligatedinto Ndel/Spel sites of pET14b pET14b-fucO pET14b-fucO PCR product offucO This study ligated into Ndel/Spel sites of pET14b pET14b-GFP-SsrApET14b-GFP-SsrA PCR product of gfp-ssrA This study ligated intoNdel/Spel sites of pET14b pET14b-D18-gldA pET14b-D18-His-gldA Ndel/Spelfragment of This study pET14b-gldA ligated into Ndel/Spel sites ofpET14b-D18 pET14b-D18-dhaK pET14b-D18-His-dhaK Ndel/Spel fragment ofThis study pET14b-dhaK ligated into Ndel/Spel sites of pET14b-D18pET14b-D18-mgsA pET14b-D18-His-mgsA Ndel/Spel fragment of This studypET14b-mgsA ligated into Ndel/Spel sites of pET14b-D18 pET14b-D18-fucOpET14b-D18-His-fucO Ndel/Spel fragment of This study pET14b-fucO ligatedinto Ndel/Spel sites of pET14b-D18 pET14b-D18-GFP-SsrA pET14b-D18-His-Ndel/Spel fragment of This study GFP-SsrA pET14b-GFP-SsrA ligated intoNdel/Spel sites of pET14b-D18 pET14b-P18-gldA pET14b-P18-His-gldANdel/Spel fragment of This study pET14b-gldA ligated into Ndel/Spelsites of pET14b-P18 pET14b-P18-dhaK pET14b-P18-His-dhaK Ndel/Spelfragment of This study pET14b-dhaK ligated into Ndel/Spel sites ofpET14b-P18 pET14b-P18-mgsA pET14b-P18-His-mgsA Ndel/Spel fragment ofThis study pET14b-mgsA ligated into Ndel/Spel sites of pET14b-P18pET14b-P18-fucO pET14b-P18-His-fucO Ndel/Spel fragment of This studypET14b-fucO ligated into Ndel/Spel sites of pET14b-P18pET14b-P18-GFP-SsrA pET14b-P18-His- Ndel/Spel fragment of This studyGFP-SsrA pET14b-GFP-SsrA ligated into Ndel/Spel sites of pET14b-P18pML-1 pET14b-His-gldA-His- Xbal/EcoRI fragment This study fucO frompET14b-His-fucO ligated into Xbal/EcoRI sites of pET14b-His- gldA pML-2pET14b-P18-gldA- Xbal/EcoRI fragment This study D18-fucO frompET14b-D18-His- fucO ligated into Spel/EcoRI sites ofpET14b-P18-His-gldA pML-3 pET14b-His-dhaK- Xbal/HindIII fragment Thisstudy His-mgsA from pET14b-His-mgsA ligated into Spel/HindIII sites ofpET14b-His- dhaK pML-4 pET14b-P18-dhaK- Xbal/HindIII fragment This studyD18-mgsA from pET14b-D18-His- mgsA ligated into Spel/HindIII sites ofpET14b-P18-His-dhaK pML-5 pET14b-His-dhaK- Xbal/Clal fragment from Thisstudy His-mgsA-His-gldA- pML-3 ligated into His-fucO Spel/Clal sites ofpML-1 pML-6 pET14b-P18-dhaK- Xbal/Clal fragment from This studyD18-mgsA-P18-gldA- pML-4 ligated into D18-fucO Spel/Clal sites of pML-2

Overexpression and Purification of Recombinant Protein

BL21*(DE3) pLysS competent cells were transformed with a plasmidcontaining the gene(s) of interest. 1 L of LB supplemented withampicillin (100 mg/L) in baffled flasks was inoculated from an overnightstarter culture. The cultures were grown at 37° C. with shaking for 7hours; protein production was induced by the addition of IPTG to a finalconcentration of 400 μM. The cultures were then incubated overnight at19° C. with shaking. Cells were harvested by centrifugation at 3320×gfor 15 minutes at 4° C., pellets were resuspended in 20 mM Tris-HCl, pH8.0, 500 mM NaCl, 5 mM Imidazole. Cells were lysed by sonication andcell debris removed by centrifugation. Recombinant protein was thenpurified from the soluble fraction by immobilized metal ion affinitychromatography.

Activity Assays

Glycerol Dehydrogenase

The activity of GldA for the oxidation of glycerol to dihydroxyacetonewas measured by following the initial rate at 340 nm for the reductionof NAD+ to NADH. Activity assays were carried out in 1 ml reactionscontaining 0.1 M potassium phosphate buffer pH 8.0, 500 μM NAD+, 2 mMMgCl₂ and 200 nM GldA. The activity of GldA for the reductionmethylglyoxal to lactaldehyde, was measured by following the initialrate of the oxidation of NADH to NAD+ at 340 nm. Activity assays werecarried out in 1 ml reactions containing 0.1 M potassium phosphatebuffer pH 8.0, 0.1 mM NADH, 2 mM MgCl2, 200 nM GldA.

Dihydroxyacetone Kinase

The activity of DhaK for the conversion of dihydroxyacetone todihydroxyacetone phosphate was measured in a coupled reaction withGlyceraldehyde 3-phosphate dehydrogenase (G3PDH) by following theoxidation of NADH to NAD+ at 340 nm. Activity assays were carried out in1 ml reactions containing 50 mM Tris-HCl, 100 mM NaCl, 1 mM ATP, 0.1 mMNADH, 2.5 mM MgCl₂, 7.2 U G3PDH, 125 nM DhaK.

Methylglyoxal Synthase

The activity of MgsA was monitored in a colorimetric assay over a timecourse. 25 μl 0.5 mM MgsA was incubated in a reaction mixture containing400 μl 50 mM imidazole pH 7.0, 25 μl 15 mM dihydroxyacetone phosphate,50 μl dH2O, the reaction mixture was incubated at 30° C. with shaking.At time intervals 50 μl of the reaction mixture was removed and added toa detection mixture containing 450 μl dH2O, 165 μl 0.1%2,4-Dinitrophenylhydrazine hydrochloric acid solution. The detectionmixture was incubated at 30° C. with shaking for 15 minutes. 835 μl of10% (w/v) NaOH was added to the detection mixture which was incubated atroom temperature for 15 minutes. Absorbances were then measured at 550nm.

1,2-Proanediol Oxidoreductase

The activity of FucO was determined for the NADH dependant reduction ofglycolaldehyde to elthylene glycol was measured by following the initialrate of the oxidation of NADH to NAD+ at 340 nm. Activity assays werecarried out in 1 ml reactions containing 100 mM Hepes, 10 μM NADH, 100μM MnCl2, 200 nM FucO.

Embedding of Strains for TEM Analysis

50 ml of LB was inoculated with one colony and grown at 37° C. withshaking to an OD600 of ˜0.4, cells were harvested by centrifugation at3000×g for 10 minutes. The cell pellet was resuspended in 2 ml 2.5%Glutaraldehyde in 100 mM cacodylate pH 7.2 and incubated for 2 hourswith gentle spinning. Cells were pelleted by centrifugation at 6000×gfor 2 minutes and were washed twice with 100 mM cacodylate pH 7.2. Cellswere stained with 1% osmium tetroxide in 100 mM cacodylate pH 7.2 for 2hours and subsequently washed twice with dH2O. Cells were dehydrated byincubation in an ethanol gradient, 50% EtOH for 10 minutes, 70% EtOHovernight followed by two 10 minute washes in 100% EtOH. Cells were thenwashed twice with propylene oxide for 15 minutes. Cell pellets wereembedded by resuspention in 1 ml of a 1:1 mix of propylene oxide andAgar LV Resin and incubated for 30 minutes with spinning. Cell pelletswere washed twice in 100% Agar LV resin. The cell pellet was resuspendedin fresh resin and transferred to a 0.5 ml mould, centrifuged for 5minutes at 3000×g to concentrate the cells to the tip of the mould andincubated for 16 hours at 60° C. to polymerise.

Sectioning and Visualisation of Samples

Samples were thin sectioned on a RMC MT-XL ultramicrotome with a diamondknife (diatome 45°) sections were placed on 300 mesh copper grids. Gridswere stained by incubation in 4.5% uranyl acetate in 1% acetic acidsolution for 1 hour followed by 2 washes in dH₂O. Grids were thenstained with 0.1% lead citrate for 8 minutes followed by a wash in ddH₂O

Electron microscopy was performed using a JEOL-1230 transmissionelectron microscope.

Culture Medium and Conditions for 1,2-Propanediol Production

The culture medium designed by Neidhardt et al., 1974 was supplementedwith 30 g/L glycerol, 10 g/L tryptone, 5 g/L yeast extract andappropriate antibiotics. Strains were cultured in sealed serum bottleswith a working volume of 100 ml at 28° C. with shaking. Cultures wereinoculated from starter cultures to starting OD₆₀₀ of 0.05. Duringgrowth 1 ml samples were removed at 0, 6, 12, 24, 48, 72 and 96 hours.

Western Blot Analysis

Nitrocellulose membranes following transfer and blocking were incubatedin primary antibody (mouse anti-GFP) followed by incubation in asecondary coupled antibody (Anti-mouse IgG AP). Bands were visualised byincubation in substrate 5-Bromo-4-chloro-3-indolyl phosphate/Nitro bluetetrazolium (BCIP/NBT).

Analysis of 1,2-Propanediol Production

In-vivo 1,2-propanediol production was determined by GC/MS analysis ofthe growth medium at time intervals (0, 6, 12, 24, 48, 72 and 96 hours).The supernatant after centrifugation, was boiled for 10 minutes at 100°C. followed by centrifugation at 19,750×g. The sample was then acidifiedwith trifluoroacetic acid to a final concentration of 0.01% followed bya second centrifugation at 19,750×g. The supernatant followingcentrifugation was diluted 1:4 in acetonitrile for GC/MS analysis.

Visualisation of Engineered Strains

Embedding of Strains for Immunolabeling

Strains were grown as described previously (“Culture medium andconditions for 1,2-propanediol production”, above) overnight, cells wereharvested by centrifugation for 10 minutes at 3000×g. The cell pelletwas resuspended in 2% formaldehyde, 0.5% gluteraldehyde in 100 mM sodiumcacodolate buffer pH 7.2 and incubated for 2 hours with gentle spinning.Cells were pelleted by centrifugation at 6000×g for 2 minutes and werewashed twice with 100 mM sodium cacodylate pH 7.2. Cells were dehydratedby incubation in an ethanol gradient, 50% EtOH for 10 minutes, 70% EtOHfor 10 minutes, 90% EtOH for 10 minutes, followed by three 15 minutewashes in 100% EtOH. Cell pellets were then resuspended in 2 ml LR whiteresin and incubated overnight with spinning at room temperature afterwhich the resin was changed and incubated for a further 6 hours. Cellpellets were resuspended in fresh resin and transferred to 1 mlembedding tubes and centrifuged at 4000×g to pellet the cells at the tipand incubated for 24 hours at 60° C. to polymerize.

Samples were thin sectioned on a RMC MT-XL ultramicrotome with a diamondknife (diatome 45°) sections were placed on 300 mesh gold grids.

Immunolabeling of Sections

Grids were equilibrated in one drop of TBST (20 mM Tris-HCl pH 7.2, 500mM NaCl, 0.05% Tween (RTM) 20, 0.1% BSA) before being transferred into adrop of 2% BSA in TBST and incubated at room temperature for 30 minutes.Grids were then immediately transferred into primary antibody (Anti-His)and incubated for 1 hour. Grids were washed in a fresh drop of TBSTfollowed by washing in a stream of TBST. Grids were equilibrated in adrop of secondary antibody (Goat anti-mouse IgG 10 nm) then incubatedfor 30 minutes in a fresh drop. Excess antibody was removed by washingin two drops of TBST before washing in a stream of ddH₂O and dried.

Staining

Grids were stained for 15 minutes in 4.5% uranyl acetate in 1% aceticacid solution followed by 2 washes in dH₂O. Grids were then stained with0.1% lead citrate for 3 minutes followed by a wash in ddH₂O.

Electron microscopy was performed using a JEOL-1230 transmissionelectron microscope.

Results

Effect of Fusing Enzymes to BMC-Targeting Peptides on Enzyme SpecificActivities

The effect of fusing either of the two targeting peptides P18 and D18 toheterologous enzymes on the functionality of those enzyme had notpreviously been investigated in detail. In this study the enzymesinvolved in the microbial synthesis of 1,2-propanediol from glycerol,namely glycerol dehydrogenase (GldA), dihydroxy acetone kinase (DhaK),methylglyoxal synthase (MgsA) and 1,2-propanediol oxidoreductase (FucO)were cloned with both N-terminal targeting peptides (P18 or D18)followed by a hexa-histidine tag. Proteins of interest were purified byIMAC and the kinetic parameters of each of the protein fusions weresubsequently determined and compared to enzymes containing only theN-terminal hexa-histidine tag. In this Example, the term “taggedproteins” refers to P18-his and D18-his containing proteins, while thehis-only containing proteins as referred to as “untagged” proteins.

It was found that the targeting peptides effect the specific activitiesof some of the proteins studied (FIG. 2). For instance, tagging GldA(the enzyme that catalyses the oxidation of glycerol) with the D18targeting peptide resulted in a reduction of its specific activity by90% compared to un-tagged GldA. Tagging GldA with the P18 targetingpeptide reduced the activity of the protein by approximately half (55%reduction) compared to un-tagged GldA (FIG. 2A). GldA's ability toreduce methylglyoxal to lactaldehyde was similarly affected, with D18having the greatest negative effect (83% reduction compared to un-taggedGldA) and P18 causing a loss of 53% of specific activity compared tountagged GldA. The activity of DhaK for the ATP dependantphosphorylation of dihydroxyacetone phosphate was determined by acoupled reaction involving a second enzyme, glyceraldehyde 3-phosphatedehydrogenase, in excess. In contrast to GldA, kinetic analysis of DhaKfused with either a P18 or D18 targeting peptide had no significanteffect on the enzyme's activity (FIG. 2B).

Tagging MgsA with either a P18 or D18 targeting peptide had a negativeeffect on enzyme activity, reducing the activity by 18% and 15%respectively in comparison to untagged MgsA, as shown in FIG. 2C. Whenfused to D18, FucO's specific activity decreased by 58% in comparison tountagged FucO and when fused to P18, FucO's specific activity decreasedby 76% in comparison to untagged FucO (FIG. 2D).

It is concluded that the fusion of targeting peptides to the N-terminiof proteins is likely to have an effect on the specific activities of asignificant proportion of said proteins. Without wishing to be bound bytheory, the inventors consider this is most likely due to changes instructural and chemical properties and potential changes in proteinfolding as a result of the fusion.

Targeting Peptides Cause Protein Aggregation that can be Visualised byTEM

The production levels and solubility of GldA, DhaK, MgsA and FucO withand without targeting peptides fused thereto were investigated bysubjecting samples of the purification process, including the solubleand insoluble fractions after clarification of the crude cell lysate aswell as the final purified protein samples, to denaturing polyacrylamidegel analysis (data not shown). DhaK and MgsA were well produced andsoluble irrespective of the presence of a P18 or D18 tag. The solubilityof FucO was not affected by targeting peptides, but the yield ofun-tagged FucO appeared slightly lower compared to FucO containing thetargeting peptides. In contrast, although, both P18 and D18-tagged GldAappeared to be produced, the protein bands were predominantly detectedin the insoluble fractions of the SDS gels.

This suggests that the fusion proteins D18-GldA and P18-GldA wereaggregating compared to GldA. P18-GldA was also found to be eluted fromthe IMAC column with an additional band of smaller molecular weight,indicative of protein degradation.

In order to investigate the aggregation behaviour of the tagged proteinsfurther, the most active protein fusions (P18-GldA, P18-DhaK, D18-MgsAand D18-FucO), the candidates for the construction of the1,2-propanediol production pathway targeted to microcompartments, werechosen to be visualised by TEM.

Strains encoding each of the tagged proteins and strains encoding theun-tagged proteins were cultured overnight without induction.Subsequently, the cells were harvested, embedded in low viscosity resin,thin sectioned and visualized using TEM. For each strain 100 cells wereexamined for protein aggregation, statistical analysis of each of thestrains is shown in FIG. 3 and representative TEM micrographs werecompiled (FIG. 8).

Control strains producing un-tagged proteins (GldA, DhaK, MgsA, FucO)displayed a ‘normal’ phenotype, with only 1% of observed cellscontaining electron dense areas indicative of aggregated proteins. Incontrast, half of all observed cells (52%) producing P18-GldA showedprotein aggregates located at the pole of the cells (FIG. 8). Theaddition of the P18 targeting peptide to the N-terminus of DhaK resultedin aggregate formation in 8% of the observed cells. Fusion of the D18targeting peptide to MgsA and FucO resulted in the presence of proteinaggregates in 12% and 4% of cells respectively.

These results confirm that the fusions between the enzymes of the1,2-propanediol production pathway and targeting peptides cause proteinaggregation.

Enzymes Fused to BMC-Targeting Peptides are Recruited to BMCs

It was investigated whether fusion proteins comprising an enzyme ofinterest fused to either the P18 or D18 targeting peptide were targetedto bacterial microcompartments.

Strains co-expressing the individual genes of the 1,2-propanediolpathway (P18-gldA, P18-mgsA, D18-dhaK, D18-fucO) and the construct forempty shell formation (pLysS-PduABB′JKNU) were cultured and therecombinant microcompartments were purified as described previously(Lawrence et al., (2014) ACS Synth. Biol. 3: 454-465). Samples weretaken throughout the purification and analysed on 15% denaturingpolyacrylamide gels for the protein profile. Analysis of the resultingSDS-PAGE gels reveals that tagging each of the proteins with a targetingpeptide facilitates their co-purification with the microcompartmentproteins. This was further confirmed by kinetic assays of the finalpurified BMC fraction.

Further evidence of protein targeting to microcompartments was providedby a protease protection assay that was previously reported by Sargentet al., (2013) Microbiology 159: 2427-2436.

Plasmids were constructed containing GFP fused to an N-terminal P18 orD18 tag and a C-terminal SsrA proteolysis tag (AANDENYALAA*). TheC-terminal SsrA tag targets proteins for degradation by the E. coliproteases ClpAP and ClpXP. E. coli competent cells were transformed withplasmids encoding the protein fusions with and without shell proteinsand the resulting strains were cultured for 24 hours, samples were takenand run on a 15% denaturing polyacrylamide gel, adjusted to cell numberas determined by OD₆₀₀ measurements. Gels were subsequently submitted toWestern blotting using an anti-GFP primary antibody.

The results show that the co-expression of GFP-SsrA fused to targetingpeptides and produced with shell proteins have the highest amount of GFP(FIG. 4 lane 7+8). In the absence of a targeting peptide GFP iseffectively degraded as represented by only a faint band present in lane6 of FIG. 4. In the absence of shell proteins, all GFP fusion proteinsare present to a much lesser extent than fusion proteins in the presenceof microcompartments. The faint bands seen could be a result of proteinaggregation, which would protect the GFP fusions from proteolyticcleavage. The band seen in lane 1 of FIG. 4 (shell only) most likelyrepresents unspecific binding of the antibody. The difference seen inband intensity between lanes 3 and 6 of FIG. 4 is likely due todifferences in expression levels as a result of the co-expression ofshell proteins.

These results are consistent with microcompartments providing protectionfrom cytosolic proteases for proteins internalised therein.

Construction of 1,2-Propanediol Producing Strains and ComparativeAnalysis of Bacterial Growth

For the in vivo production of 1,2-propanediol, single plasmids wereengineered by using link and lock cloning combining firstly the genescoding for the most active protein fusions (pML-6 containingP18-his-gldA, P18-his-dhaK, D18-his-mgsA, D18-his-fucO) and secondly thesame genes but without targeting sequences (pML-5 containing his-gidA,his-dhaK, his-mgsA, his-fucO). Both plasmids were used to transform theE. coli strain BL21*(DE3).

With the aim of targeting the 1,2-propanediol producing enzymes torecombinant microcompartments, strains were engineered to co-express the1,2-propanediol production plasmids with the genes coding for theprotein shell (pLysS-PduABB′JKNU). The shell protein construct allowsfor the formation of a microcompartment shell to which the fusionenzymes are recruited by virtue of their BMC-targeting peptide.Additionally, the following control strains were set up: firstlyBL21*(DE3) transformed with pET14b and pLysS; and secondly, a shell onlystrain transformed with pET14b and pLysS-PduABB′JKNU. All strains werecompared for the production of 1,2-propanediol.

The culture medium designed by Neidhardt et al., 1974 (Neidhardt F C,Bloch P L, Smith D F. Culture Medium for Enterobacteria. Journal ofBacteriology. 1974; 119(3):736-747) was supplemented with 30 g/Lglycerol, 10 g/L tryptone and 5 g/L yeast extract and appropriateantibiotics. Strains were cultured in sealed serum bottles with aworking volume of 100 ml at 28° C. with shaking The cultures werestarted with an initial OD₆₀₀ of 0.05 by inoculation from 5 ml startercultures. During growth, 1 ml samples were collected at 0, 6, 12, 24,48, 72 and 96 hours and optical densities at 600 nm were measured. Theresulting growth curves (not shown) indicate that strains encodingproteins with targeting peptides (either with shell proteins or without)grow slower and reach a lower final optical density in comparison tostrains expressing un-tagged proteins and control strains. Furthermore,cell densities declined from 24 hours in strains producing un-taggedenzymes whereas the cell densities of the strains with tagged enzymesremained constant, which indicates that cells with tagged enzymes cellsare being protected from a toxic intermediate.

In Vivo 1,2-Propanediol Production is Elevated in Strains ProducingEnzymes with Targeting Sequences

The 1,2-propanediol content in the growth media of the various strainswas quantified by gas chromatography-mass spectrometry (GC-MS). Wholecell samples were collected at 0, 6, 12, 24, 48, 72 and 96 hours and thesupernatant following centrifugation was prepared for GC-MS analysis asdescribed in materials and methods. The measured 1,2-propanediol contentas shown in FIG. 5 is expressed for a cell density of OD₆₀₀=1. FIG. 6shows the measured 1,2-propanediol content not adjusted for a celldensity of OD₆₀₀=1.

Strains encoding un-tagged 1,2-propanediol producing enzymes with andwithout shell proteins showed low 1,2-propanediol production despitegrowing well and reaching the highest cell densities at 96 hours. Bothstrains reached the maximum product concentration (at 96 hours) of 3.59mM/OD₆₀₀=1 in the absence of shell proteins and 1.95 mM/OD₆₀₀=1 in thepresence of shell proteins.

The highest product concentrations were detected in the growth media ofstrains producing proteins tagged with targeting peptides. Although bothof these strains (with and without shell proteins) grew to lower densitythan the strains harbouring un-tagged proteins, and despite the negativeeffect the targeting peptide has on the specific activities of theindividual enzymes, they produced significantly more 1,2-propanediol(FIG. 5) than the strains lacking the targeting peptides. The straincontaining tagged 1,2-propanediol enzymes and shell proteins reached afinal yield of 7.10 mM/OD₆₀₀=1. However, the highest final yield of11.56 mM/OD₆₀₀=1 was observed when the shell proteins were not present.1,2-propanediol was not detected in control stains (wild type E. coliand a strain producing shell proteins only).

A comparison of FIG. 5 and FIG. 6 shows that the increase in1,2-propanediol production levels is not simply an effect of differencesin cell densities i.e. even though the strains with tagged enzymes don'tgrow as well as the strains without tagged enzymes, they still producemore 1,2-propanediol.

The higher product yield exhibited by the strain producing taggedenzymes in the absence of shell proteins was unexpected.

To investigate if aggregation of the proteins were causing this effect,electron microscopy and immunolabeling were used to visualise thesubcellular organisation and location of the recombinant proteins in thevarious strains. Sections of the strains were labelled withanti-histidine primary antibody designed to bind to the hexa-histidinetag on the N-terminus of proteins in our pathway thereby revealing theintracellular location. A secondary antibody conjugated to 10 nm goldparticles was used to bind to the primary antibody thereby revealing theintracellular location of 1,2-propanediol producing enzymes.

Control strains (wild type and shell only) showed a small amount ofantibody binding around the membrane of the cells (FIGS. 7A and 7B);this is likely due to unspecific binding. Aggregates are visible inapproximately 100% of observed cells expressing P18/D18-tagged proteins,regardless of the presence or absence of shell proteins, and it is inthese areas that the vast majority of antibody binding occurs (FIGS. 6Cand D). Such structures cannot be seen in cells expressing un-taggedenzymes (FIGS. 6E and F), suggesting that it is the presence of thetargeting peptides that facilitates the aggregation of proteins.

It is concluded that aggregation occurs due to tagging proteins ofinterest with targeting peptides and it is this aggregation that resultsin a significant increase in product yield despite the reduction inspecific activities of the individual tagged pathway enzymes.

It has been determined that the fusion of BMC-targeting peptides to theindividual enzymes in the pathway for the production of 1,2-propanediollowers the specific activities of the enzymes in some (most) cases. Thesolubility of each enzyme was also affected to varying degrees, withGldA forming large inclusion bodies in the majority of cells observed byTEM when fused with a targeting peptide compared to un-tagged GldA. Ithas also been demonstrated that the addition of a BMC-targeting tagrecruited the enzymes to BMCs and that purified samples thereof remainedmetabolically active.

Despite the significant decrease of enzyme activity seen with theaddition of targeting peptides to both GldA and FucO, expression of thecomplete tagged pathway enzymes led to an increase in product formationas compared to strains in which untagged enzymes were expressed. Ratherunexpectedly, the presence of the microcompartment shell was notrequired for the increased product formation and, furthermore, thestrain generating the most 1,2-propanediol produced tagged1,2-propanediol pathway enzymes, but no shell proteins. This strainshowed an increase in product formation of 245% OD-adjusted incomparison to the strain producing un-tagged enzymes; despite the lowerin vitro activity of the individual tagged proteins compared to theun-tagged proteins.

TEM analysis showed that co-production of all four tagged enzymesresulted in protein aggregation and deposition at the poles of nearlyall cells observed and it is this aggregation that appears to provide asignificant benefit to the efficiency of the pathway. Aggregation of ourproteins of interest is likely due to the amphipathic helical nature ofthe BMC-targeting sequences and/or by their coiled coil structure.Without wishing to be bound by theory, it can be considered that theaggregation creates a scaffolding effect that result in increasedchanneling of substrates and products between enzymes, similarly to theenvironment inside a microcompartment.

This study is the first to demonstrate that the presence of shorttargeting peptides can not only convert individual fusion proteins butalso whole pathways into active aggregates that allow for increasedproduct yield in vivo. These aggregations of multiple enzymes allow forincreased localised concentrations of enzymes and intermediates andpossibly channeling between them thereby resulting in a higher productyield. This is the first study to demonstrate that increased productyields can result from tagging enzymes in a metabolic pathway for theproduction of said product with a BMC targeting sequence in a celllacking BMCs themselves.

1. A genetically modified microorganism comprising one or moreheterologous nucleic acid molecules together encoding at least threedifferent proteins, each protein comprising an enzymatic domain and abacterial microcompartment-targeting signal polypeptide, wherein saidenzymatic domains each catalyse a different substrate to productconversion in the same metabolic pathway, and wherein said microorganismis essentially free of bacterial microcompartments (BMCs).
 2. Thegenetically modified microorganism of claim 1, wherein saidmicroorganism comprises one or more heterologous nucleic acid moleculestogether encoding at least four different proteins, each proteincomprising an enzymatic domain and a bacterialmicrocompartment-targeting signal polypeptide, wherein said enzymaticdomains each catalyse a different substrate to product conversion in thesame metabolic pathway.
 3. The genetically modified microorganism ofclaim 1, wherein each of said heterologous nucleic acid moleculesencodes only one of said proteins.
 4. The genetically modifiedmicroorganism of claim 1, wherein each protein is over-expressedrelative to the level of expression of said protein in a microorganismof the same strain which lacks said one or more heterologous nucleicacid molecules.
 5. The genetically modified microorganism of claim 1,wherein said microorganism comprises aggregates comprising said at leastthree proteins.
 6. The genetically modified microorganism of claim 1,wherein each protein comprises an enzymatic domain and a BMC-targetingsignal polypeptide linked by an amino acid linker, preferably whereinsaid amino acid linker lacks stable secondary structure.
 7. Thegenetically modified microorganism of claim 1, wherein eachBMC-targeting signal polypeptide comprises an amphipathic alpha helix.8. The genetically modified microorganism of claim 1, wherein eachBMC-targeting signal polypeptide comprises the following amino acidsequence: X₁X₂X₃X₄X₅X₆X₇X₈X₉, wherein: X₁, X₄, X₅, X₈, and X₉ are eachindependently hydrophobic amino acids selected from the group consistingof I, L, V, M, F, Y, A and W; X₂, X₃ and X₆ are each independently polaror charged amino acids selected from the group consisting of Q, N, T, S,C, D, E, R, K and H; and X₇ is any amino acid.
 9. The geneticallymodified microorganism of claim 1, wherein each BMC-targeting signalpolypeptide comprises a sequence selected from the group consisting ofLEQIIRDVL (SEQ ID NO:1), LETLIRTIL (SEQ ID NO:2), LETLIRNIL (SEQ IDNO:3), LRQIIEDVL (SEQ ID NO:4), IEEIVRSVM (SEQ ID NO:5), IEQVVKAVL (SEQID NO:6), VEKLVRQAI (SEQ ID NO:7), IQEIVRTLI (SEQ ID NO:8), VEEIVKRIM(SEQ ID NO:9), IESMVRDVL (SEQ ID NO:10), VQDIIKNVV (SEQ ID NO:11),IRQVVQEVL (SEQ ID NO:12), VRSVVEEVV (SEQ ID NO:13) and ARDLLKQIL (SEQ IDNO:14) or a variant sequence thereof.
 10. The genetically modifiedmicroorganism of claim 1, wherein said microorganism is a bacterium. 11.The genetically modified microorganism of claim 1, wherein saidmicroorganism does not naturally comprise the genes necessary for theexpression of BMCs.
 12. The genetically modified microorganism of claim1, wherein said microorganism is a strain that naturally comprises thegenes necessary for the expression of BMCs, and wherein expression ofsaid BMCs is inducible by the presence of one or more inducer molecules,but wherein said microorganism is present in a culture medium in whichthe level of said inducer molecule(s) is too low to induce theexpression of said BMCs.
 13. The genetically modified microorganism ofclaim 1, wherein said microorganism is a strain that naturally comprisesthe genes necessary for the expression of BMCs but wherein saidmicroorganism comprises a loss of function mutation in one or more ofsaid genes.
 14. The genetically modified microorganism of claim 13,wherein said microorganism comprises a loss of function mutation in atleast one gene encoding a protein comprising a BMC domain and in atleast one gene encoding a protein comprising a bacterialmicrocompartment vertex domain.
 15. The genetically modifiedmicroorganism of claim 13, wherein said microorganism comprises amutation in the regulatory region of the BMC operon(s), preferably inthe operon's promoter.
 16. A method of producing a product of interest,said method comprising growing the genetically modified microorganism ofclaim 1 under conditions wherein the product is produced and optionallyrecovering the product.
 17. A cell free system comprising aggregatescomprising at least three different proteins, each protein comprising anenzymatic domain and a bacterial microcompartment-targeting signalpolypeptide, wherein said enzymatic domains each catalyse a differentsubstrate to product conversion in the same metabolic pathway, andwherein said system does not comprise bacterial microcompartments.
 18. Amethod of producing a product of interest, said method comprising: i)providing a cell free system comprising aggregates comprising at leastthree different proteins, each protein comprising an enzymatic domainand a bacterial microcompartment-targeting signal polypeptide, whereinsaid enzymatic domains each catalyse a different substrate to productconversion in the same metabolic pathway for the production of theproduct of interest, and wherein said system does not comprise bacterialmicrocompartments; ii) applying to said system the substrate of thefirst substrate to product conversion in the metabolic pathway that iscatalysed by one of said enzymatic domains; and iii) optionallyrecovering the product of interest.
 19. The method of claim 18, whereinsaid aggregates are obtained from a cell lysate of the microorganism ofclaim 1.